Fig. 5. Electrophoretic mobility shift assay results for the AZRE. (A) Sequence-specific interaction of nuclear proteins with the AZRE. The 23 bp AZRE containing a fragment between nucleotides –190 and –168 relative to the transcription start site of Oshsp17.3 was radiolabelled, and 1 ng of the probe was incubated with 10 µg of the nuclear proteins prepared from rice etiolated seedlings under AZC treatment for 6 h. The positions of the DNA–protein complexes are shown as C1 and C2 by arrows. The free probe is indicated with an asterisk (*). (B) Effect of CHX on the interaction of nuclear proteins with the AZRE. Three-day-old rice seedlings were treated with CHX (2 µg ml⁻¹) and with (left) or without (right) AZC for 6 h. (C) Cross-competition analysis of the HSE. The 23 bp HSE-containing fragment located at –87 to –65 relative to the transcription start site of Oshsp17.3 was used as a competitor. (D) Stress-dependent interaction of nuclear proteins with the AZRE. Nuclear extracts were prepared from seedlings treated with AZC, As, or HS for 2 h and by 28 °C [control (C)] or Cd for 6 h.

Specificity of nuclear proteins interacting with the 9 bp AZRE in vitro by EMSA. To characterize the putative AZRE further, EMSA was used to determine whether it can interact with trans-acting factor(s) present in nuclear extracts. A 23 bp synthetic oligonucleotide encompassing the AZRE was used as a probe and was labelled with 32P by T4 polynucleotide kinase. Then, the probe was incubated with nuclear extracts from AZC-treated rice seedlings. Two DNA-bound protein bands (C1 and C2) were detected on the gel (Fig. 5A). The intensity of C1 appeared to be stronger than that of C2, and C1 could be further competed out by a 200-fold excess of AZRE probe without a 32P label. In addition, nuclear extracts from seedlings treated with AZC plus CHX for 6 h or CHX alone did not affect the levels of C1 and C2 interacting with the AZRE (Fig. 5B), which suggests that de novo synthesis of proteins is not required for the interaction. Because the 9 bp AZRE is involved in the HSR, it was also tested whether the 9 bp AZRE could be cross-competed by an HSE. This 23 bp HSE probe encompasses a perfect HSE (CTTCTAGAAACTTCC). Interestingly, the C2 but not the C1 band could be competed out by a 200-fold excess of HSE without being 32P labelled (Fig. 5C). Nevertheless, these results show that the AZRE can be specifically recognized by the nuclear proteins from AZC-treated rice seedlings. Because the AZRE was required for the response to HS and AZC, EMSA was used to examine whether nuclear extracts from rice seedlings treated with different inducers can bind to the AZRE. In nuclear extracts from seedlings treated with As (2 h), Cd (6 h), HS (2 h), or AZC (2 h), the two DNA–protein complexes C1 and C2 were also detected in all extracts, including control extracts (Fig. 5D). Additionally, the C1 band in the control was competed out by a 10-fold amount of the AZRE (Fig. 5D, first left lane). The intensity of the C1 complex with AZC treatment was found to be \~5-fold higher than that of the control by measuring the integrated density with Scion image software. However, the intensity of the C2 band in 2 h AZC-treated and control nuclear extracts was lower than that in extracts receiving 6 h AZC, As, Cd, and HS treatments. Nevertheless, the results demonstrate that nuclear proteins from rice seedlings bind to the AZRE in a stress-dependent manner and depend on the duration of AZC treatment. Deletion of the AZRE on the promoter of Oshsp17.3 can reduce OsHsfA4b-modulated activation. According to the indispensable role of the AZRE for HS responsiveness (Fig. 3) and the results of EMSA (Fig. 5C), it was speculated that the AZRE can be specifically recognized by HSFs. Thus, tests were conducted to determine whether the AZRE was necessary for the interaction between OsHsfs and the promoter of Oshsp17.3. According to the expression analysis of 23 rice HSF genes, OsHsfA4b (Os01g54550) and OsHsfB4b (Os07g44690) were found to be induced by AZC treatment (data not shown). Two effector constructs, pHsfA4b and pHsfB4b, under the control of the maize constitutive Ubi1 promoter, were made (Fig. 6A). GUS activity in coleoptiles co-transformed with one reporter (p567 or p567DAZRE) and one effector (pHsfA4b or pHsfB4b) construct at a ratio of 1:1 was compared. As shown in Fig. 6B, when co-transformed with pHsfA4b, p567 achieved 2.8-fold induction in relative GUS activity but p567DAZRE achieved only a 1.8-fold induction. However, co-transformation of pHsfB4b was not significantly induced on p567 or p567DAZRE. To confirm these results further, the p631 reporter construct was also co-bombarded with pHsfA4b or pHsfB4b and no effect on the non-AZC-inducible Oshsp16.9A promoter was found (1.3- and 1.5-fold induction, respectively) (Fig. 6B). Also, transient expression of another highly AZC-inducible HSF gene, OsHsfC1b (Os01g53220), had no effect on the activation of Oshsp17.3 and Oshsp16.9A (data not shown). Thus, HSFs are possibly an integral part of the AZRE-dependent induction pathway of sHSP-CIs. The differential transcription of sHSP genes may have resulted from the action of different kinds of HSFs or additional transcription factors responding to environmental stresses (Rojas et al., 2002). Two DNA–protein complexes (C1 and C2) were observed by EMSA. Both of the interaction complexes can be competed out by cold AZRE, which indicates that the AZRE–protein interactions are sequence specific and may involve two distinct trans-acting proteins (Fig. 5A). Also, nuclear extracts from untreated AZRE (Fig. 5D), and the level of bound proteins was not affected by CHX (Fig. 5B). These data show that de novo protein synthesis was not required for the trans-acting factors recognizing the AZRE, and thus these factors constitutively exist in the cells. However, nuclear extracts prepared from seedlings treated for 6 h with AZC, Cd, As, or HS showed a lower intensity of C1 and C2 than those from control seedlings (Fig. 5D), which suggests that the AZRE may interact with nuclear proteins in a stress-dependent manner. However, whether trans-acting factor(s) directly sense AZC or are only indirectly activated by AZC needs further study. Interestingly, it was also found that the AZRE was indispensable for HS activation of Oshsp17.3 and Oshsp18.0 in the transient expression system (Fig. 3), that the C2 band can be competed out by the HSE (Fig. 5C), and that HS strongly boosted both C1 and C2 signals (Fig. 5D). These data are in agreement with the observation of Sugio et al. (2009), who showed HSFs to be involved in the AZC response in Arabidopsis. Among the HSFs identified in rice, OsHsfA4b, OsHsfB4b, and OsHsfC1b show high AZC responsiveness (data not shown). Co-expression of OsHsfA4b in rice coleoptiles could activate both p567 and p567DAZRE constructs in the absence of AZC (Fig. 6B), but the induction level in p567 was 67% higher than that in p567DAZRE. In contrast,


Figure 1. SDHB expression is high in CRC cell lines. (A) Quantitative RT-PCR analysis of SDHB mRNA expression in four colorectal cancer (CRC) cell lines compared to the normal colorectal epithelial cell line NCM460. (B) Semi-quantitative RT-PCR further confirms increased SDHB transcript levels in CRC cell lines relative to NCM460. (C) Western blotting analysis shows decreased SDHB protein expression in all four CRC cell lines compared to the normal epithelial control.

SDHB is the key enzymatic component of mitochondrial enzyme SDH constructively expressed across species, which oxidizes succinate to fumarate in the TCA cycle and feeds electrons into the mitochondrial respiratory chain for ATP production. We examined SDHB mRNA and protein levels in four CRC cell lines and normal colorectal epithelial cell line NCM460. As shown in Figure 1 both SDHB mRNA level and protein level were relatively high in all four CRC cell lines (SW480, SW620, SW1116, and HT-29), compared to non-CRC epithelial NCM460 cells.


Effects of KBrO3 on mtDNA copy number and topology. (A) Dot-blot of purified mtDNA from control cells and cells treated with 30 μM KBrO3. Oxidative damage is detected using an antibody against 8-oxodG by SouthWestern analysis and normalized against total mtDNA by Southern hybridization on a separate blot. (B) Dot-blot for antibody verification, showing control mtDNA and mtDNA treated in vitro with 7.4% H2O2 for 30 min. (C) Topology gel of mtDNA. KBrO3 treatment had no effect on mtDNA topology. 16.6, 16.6-kb linear molecules; CAT, catenanes; OC, open circles; SC, supercoiled mtDNA. (D) mtDNA copy number levels during the KBrO3 treatment time course. Despite the damage, mtDNA copy number is not unaffected by KBrO3.

Although KBrO3 did not have as dramatic an effect on nuclear DNA damage signaling as UV or H2O2, it induced a marked increase in mtDNA damage (Figure 1A). The damage did not result in copy number depletion or any effect on the supercoiling of mtDNA (Figure 1, C and D).


Figure 1. Purification of chicken FANCD2, FANCI, FANCL andhuman UBE2T. (A) Purified chicken FANCD2 and FANCI, used forin vitro assays, were analyzed by 15% SDS-PAGE with CoomassieBrilliant Blue staining. Lane 1 indicates the molecular mass markers.Lanes 2 and 3 indicate purified FANCD2 and FANCD2 K563R,respectively. Lanes 4–9 indicate purified FANCI, FANCI K525R,FANCI R1288Q, FANCI Dx6, FANCI Dx6 K525R and FANCIEx6, respectively. (B) Purified human UBE2T, used for in vitromonoubiquitylation assays, was analyzed by 15% SDS-PAGE withCoomassie Brilliant Blue staining. Lane 1 indicates the molecularmass markers. Lane 2 indicates purified UBE2T. (C) PurifiedGST-tagged chicken FANCL, used for in vitro monoubiquitylationassays, was analyzed by 15% SDS-PAGE with Coomassie BrilliantBlue staining. Lane 1 indicates the molecular mass markers. Lane 2indicates purified GST-tagged FANCL.

Chicken FANCI and FANCD3 were purified as recombinant proteins (Figure 1A, lanes 3 and 4), and the IDcomplex was prepared by mixing them in 1:1 stoichiometry. We also purified UBE2T and FANCL, which areknown as the E2 and E3 proteins for FANCD2monoubiquitylation, respectively, as recombinantproteins (Figure 1B and C).


FIGURE 6. Incorporation of single UV photoproducts increases nucleosome dynamics. A, schematic illustration of the incorporation of a single CPD or 6-4PP into the 601 sequence. The DFs are 12-mer oligonucleotides with the sequences shown. Solid triangles indicate the positions of the photoproduct. The dark circle represents Cy5, and the open circle represents Cy3. B, FRET changes for the single CPD containing NCPs. The 601 sequence was changed according to the DF and denoted 601.DF1. The left panel shows emission spectra for the CPD containing NCPs compared with undamaged NCPs. The right panel shows the FRET efficiency values determined in each case, where the value for 601.DF1 NCPs was set to 1. The values shown are the mean ± S.D. of three independent experiments. C, the same as B except the NCPs contained a single 6-4PP and the sequence denoted 601.DF2. D, relative FRET efficiency of salt dependence of NCPs containing a single CPD. The FRET efficiency at the lowest salt concentration (50 mM) was set to 1. Values are the mean ± S.D. of three independent experiments. E, salt titration profile of NCPs containing a single 6-4PP. Values were plotted the same as in D.

Taining a single CPD or 6-4PP lesion is 4.6 nm and 4.2 nm, respectively. Furthermore, in each case, the FRET efficiency is reduced, indicating that the introduction of a single UV lesion in nucleosome DNA can increase NCP unwrapping. This supports the UV dose-dependent FRET change we observed with UV-irradiated NCP DNA (Figure 3). Moreover, NCPs with either a single CPD or 6-4PP are more sensitive to salt (Figure 6, D and E). In the range of 0–1 M salt concentrations, the FRET efficiency in NCPs with single UV lesions is significantly lower than that of undamaged NCPs, indicating that NCPs containing just one DNA photoproduct are significantly less stable with increasing salt.


Figure 1. Reduced PtdCho synthesis in CCTα-deficient macrophages. (A) Wild-type (WT; n = 12) or CCTα-deficient (KO; n = 12) macrophages were labeled for 6 h with 1 μCi/ml [3H]choline, and incorporation into PtdCho was normalized to cellular protein. (B) Relative transcript levels for CCTα, CCTβ2, CCTβ3, or PEMT were determined by real-time qRT-PCR in wild-type (n = 3) or CCTα-deficient (n = 3) macrophages. (C) CCT enzyme activity in wild-type (△; n = 4) or CCTα-deficient (●; n = 4) macrophage lysates was determined in vitro as a function of protein concentration. (D) Protein expression levels of CCTα (42 kD) and CCTβ3 (39 kD) were determined in wild-type and CCTα-deficient macrophage lysates (50 μg) by immunoblotting with isoform-specific antibodies. Immunoblots are representative of macrophages from two mice of each genotype. Cells (HEK293T) transfected with expression plasmids encoding CCTα or CCTβ3 were lysed, and 5 μg of protein was loaded as a positive control. Multiple immunopositive bands in a lane correspond to different phosphorylation states of the CCT protein (Jackowski, 1994; Lykidis et al., 1998). Error bars indicate mean ± SE. *, P < 0.05; **, P < 0.01.

Mice with CCTα-null macrophages were generated as described previously (Zhang et al., 2000). Briefly, mice carrying a floxed Pcyt1a gene were crossed with mice expressing Cre recombinase under the macrophage-specific LysM promoter. Two loxP sites flanked a 12.5-kb fragment of the Pcyt1a gene containing exons 5–9 (Karim et al., 2003) and the intervening introns. Deletion of the region between the loxP sites resulted in expression of a truncated CCTα protein lacking the catalytic and regulatory domains at the carboxy terminus. Mice homozygous for the floxed gene and heterozygous for LysMCre were mated, and pups were genotyped to obtain either CCTα knockout or littermate wild-type control macrophages.Peritoneal macrophages were elicited, and the CCTα-deficient macrophage population showed increased rates of de novo phosphatidylcholine (PtdCho) synthesis measured by [?H]choline incorporation into the lipid fraction of adherent cells (Fig. 1 A). The deletion in this macrophage model was not completely penetrant (Zhang et al., 2000). Quantitative RT-PCR (qRT-PCR) analysis revealed that the CCTα transcript level was significantly increased to about 30% of wild-type levels (Fig. 1 B). The CCTα-null cells were ≥95% macrophages as determined by microscopy after staining with macrophage-specific anti–mouse CD11b. Staining for CCTα protein showed that 20–30% of macrophages isolated from knockout mice (n = 4) remained positive for expression, confirming incomplete Cre recombinase activity.Thus, residual PtdCho synthesis detected by metabolic radiolabeling in CCTα-null populations was due not only to expression of the alternate isoform CCTβ3 (Fig. 1 B) but also to the presence of cells that did not delete the CCTα gene. qRT-PCR showed that the CCTβ2 isoform was not expressed in macrophages, while CCTβ3 expression was increased in knockouts. CCTβ3 shares the primary biochemical function of CCTα and can compensate for its loss to support membrane synthesis in cultured cells (Karim et al., 2003). Previous work indicated increased CCTβ2 expression in CCTα-deficient cells (Zhang et al., 2000), but at that time, CCTβ3 had not been discovered; both isoforms share an epitope detected in earlier immunoblots (Karim et al., 2003).Total CCT enzyme activity was significantly higher in lysates from CCTα-null macrophages compared with wild-type populations (Fig. 1 C), consistent with previous studies (Zhang et al., 2000). Immunoblotting with isoform-specific antibodies revealed a decrease in CCTα protein and an increase in CCTβ3 protein reflecting mRNA changes (Fig. 1 D). Macrophages from two knockout and two wild-type mice were pooled by genotype, and immunoblots were prepared from six replicates per genotype. CCTα protein levels in the knockout population were 0.36 ± 0.08 relative to wild-type (set at 1.00). The reduction in CCTα transcript correlated with protein levels and the number of macrophages lacking CCTα expression by microscopy. These data confirm loss of CCTα expression in the majority of cells and provide the first example of adaptive regulation of CCTβ3 expression in response to PtdCho deficiency.


Figure 2. Differential inhibitory effects of SAUGI on the activities of (A) SAUDG, (B) EBVUDG, (C) HSVUDG, (D) human UDG and (E) TBUDG. Except for TBUDG, addition of SAUGI increased the specific uracil-removing activity of SAUDG and the other three UDGs in a dosage-dependent manner.

The inhibiting effects of SAUGI on five UDGs We next used uracil removal assays to investigate the ability of SAUGI to inhibit the same five UDGs that were used in the surface plasmon resonance experiments. In the absence of SAUGI, all the UDGs removed the uracil from the uracil-containing DNA substrates effectively, and the resulting apyrimidinic site was cleaved by heat and alkali (Figure 2A-E, lane 2). The addition of SAUGI reduced the cleaved products in the SAUDG, EBVUDG, HSVUDG and human UDG reactions (Figure 2A-D, lanes 3–7; Supplemental Figure S4). SAUDG, EBVUDG and HSVUDG activity was initially inhibited when the molar ratio of SAUGI to UDG reached 1:1, but a ratio of 2:1 was needed for the inhibition of human UDG activity. Interestingly, although the structure of TBUDG is conserved relative to the other four UDGs, SAUGI had no significant effect on this UDG’s activity (Figure 2E). These results correspond to those found in the BIAcore study. We therefore conclude that SAUGI has a relatively greater effect on the two viral UDGs than on human UDG. Since HSVUDG and EBVUDG have been shown to play an important role during virus replication (29), these results suggest a potential use of SAUGI as an anti-virus treatment. The inhibitory effects of SAUGI mutants on the uracil-removing activities of human and HSV UDGs In our previous study, we found phage UGI had an extremely high binding affinity to human UDG that was 12–13 times greater than that of SAUGI (16). We found here that phage UGI also inhibited both HSVUDG and human UDG more effectively than SAUGI (compare Supplemental Figure S6 with Figure 2C and D). We speculated that SAUGI’s relatively low binding affinity to human UDG and its high binding affinity to viral UDG might potentially allow it to be used to prevent viral UDG repair activity without having too great an effect on human UDG. In our next experiment, we therefore used structural-based protein engineering to further modify SAUGI’s activity on human UDG and HSVUDG.


Figure 2. HITS-CLIP reveals HIV-1 regions bound by Ago2 in HIV-1 infected cells. (A) HEK293T cells were transfected with plasmid expressing either GFP-Ago2 or control GFP. Twenty-four hours later, they were infected with VSVg-pseudotyped HIV-1 NL4-3 at a M.O.I. of 1 or mock infected. Twenty-four hours later, cells were subjected to UV cross-linking and RNA–protein complexes were subjected to immunoprecipitation using an anti-GFP antibody. IPed RNA was radiolabeled with [α-32P]ATP and samples resolved on SDS-PAGE. Radiolabeled RNA was visualized by autoradiography. RNAs migrating at a higher molecular mass than the protein of interest (GFP-Ago2:RNA, between 125 and 225 kDa) were extracted from the membrane, reverse transcribed into cDNA and subjected to high throughput sequencing. (B) Reads obtained from non-infected (NI) and infected (INF) samples were uploaded to CLIPZ software. Reads that aligned to the human genome were sorted into categories: messenger RNA (mRNA), microRNA (miRNA), ribosomal RNA (rRNA), repeat, small non-coding RNA (snRNA), small nucleolar RNA (snoRNA), transfer RNA (tRNA) and other. Pie charts correspond to the distribution of the mean value of three biological replicates. (C) Reads obtained from infected samples were aligned to the HIV-1 NL4-3 sequence. Only reads ≥50 nucleotides length that aligned once without mismatch were considered for this analysis. Reads obtained from three independent HITS-CLIP experiments are presented in shades of orange and those obtained from the corresponding inputs in shades of blue. The map of the HIV-1 genome is presented below. Splice donor (D) and acceptor (A) sites are indicated. Peaks corresponding to clusters 2, 13, 16, 21, 25 and 30 are indicated by arrows.

Immunopurified RNAs were radiolabeled, separated on SDS-PAGE and transferred to nitrocellulose membrane. Autoradiograms revealed the presence of RNA–protein complexes migrating from 100 kDa, the expected size of GFP-Ago2, to higher molecular weights corresponding to the size distribution of RNAs associated with GFP-Ago2 (Figure 2A). The levels of RNAs cross-linked to GFP-Ago2 were similar in presence or absence of infection but were highly enriched compared to the control GFP immunoprecipitation, confirming the specificity of our IP conditions. Ago2-RNA complexes migrating between 125 kDa and 225 kDa, corresponding to nucleic acids up to 300 nucleotides length, were extracted from the nitrocellulose membrane. Ago2 cross-linked RNAs were purified and reverse transcribed to generate small cDNA Illumina libraries. cDNA ranging from 20 to 75 base pairs were purified for subsequent deep sequencing. Libraries were also generated from GFP control samples. However, the levels of cDNA obtained from these samples were not sufficient for subsequent deep sequencing. High throughput sequencing was conducted on small RNA libraries generated from six independent Ago2-GFP HITS-CLIP experiments prepared from either uninfected or HIV-1 NL4-3 infected cells and their corresponding inputs. HITS-CLIP experiments yielded between 15 and 24 million sequencing reads and inputs between 17 and 30 million sequencing reads (Table 1).Ago2 HITS-CLIP analysisTo assess the quality of our HITS-CLIP experiments, we first uploaded our sequencing reads to the CLIPZ software, specifically dedicated to HITS-CLIP data analysis (44). The number of distinct reads mapping to the human genome and the quality of the sequences were similar to other Ago2 HITS-CLIPs analyzed with the CLIPZ tool (data not shown). The alignment of reads in the input and in the HITS-CLIP experiments showed that whereas rRNAs, tRNAs and mRNAs were the predominant reads in the input (45%, 12% and 17%, respectively), a 5-fold enrichment of mRNAs, but not of snoRNAs, snRNAs, rRNAs or tRNAs, could be observed in the HITS-CLIP (Figure 2B). We did not observe any enrichment in miRNAs likely because our HITS-CLIP procedure favored long reads over short ones. We then sought to identify Ago2 binding sites on the viral gRNA. For this, HITS-CLIP data were analyzed in two different ways. First, trimmed reads longer than 15 nucleotides were aligned to the viral genome with a maximum of one mismatch allowed. Reads (≥50 nucleotides) of the HIV-1 RNA genome (starting at the transcription start site) from the three HITS-CLIPs (in orange) and their corresponding input (in blue) were plotted (Figure 2C). As the R region is present at both ends of the viral genome, the reads could not therefore be assigned unambiguously to the 3' or 5' ends. They were arbitrarily assigned at the 3' end of the viral genome. Several HIV-1 peaks were detectable in the input but whether they correspond to small RNA produced by the virus or degradation products was unclear. In Ago2 HITS-CLIP, we identified 40 discrete Ago2 binding sites along the viral genome with an accumulation of clusters at the central region and the 3' end of the genome (Figure 2C). Both analyses of our HITS-CLIP data gave very similar results as shown by the comparison of their mean traces (Supplementary Figure S2). We focused our interest on 31 clusters of reads based on the reproducibility of the peaks in the three HITS-CLIP experiments and the enrichment of reads in the IP over the input (Supplementary Figure S3 and Supplementary Table S1).Our study focused on HIV-1 RNA regions that correspond to Ago2 binding sites by only considering reads of 50 nucleotides length. We identified more than 40 reproducible binding sites distributed along the HIV-1 gRNA (Figure 2C). Remarkably, reads from viral origin represented <1% of the total reads in inputs, a result in agreement with other studies (22,30,32). In sharp contrast, between 8 to 25% of these long reads were from viral origin in our HITS-CLIP data, confirming that viral RNAs are preferentially enriched in Ago2 co-immunoprecipitation (Figure 1 and Table 1). Two recent reports have also explored the interaction between Ago proteins and HIV-1 RNAs using similar photoactivable ribonucleoside-induced cross-linking and immunoprecipitation (PAR-CLIP) and HITS-CLIP approaches (22,32). Contrary to our study, Whisnant et al. detected very low levels of HIV-1 PAR-CLIP reads among the total of assignable reads (below 1%). This discrepancy could be due to differences in experimental conditions, as we used an overexpressed GFP-Ago2 together with stringent washing conditions in order to improve the efficiency and specificity of our immunoprecipitation conditions. Nevertheless, Whisnant et al. (22) identified a number of RISC clusters on HIV-1 RNA in infected TZM-bl HeLa and C8166 T cell lines. Importantly, our study identified 19 clusters that overlap with clusters identified in at least one of their two cell lines including cluster 2, 13, 16 and 21 (data not shown) indicating that these HIV-1 RNA regions are indeed conserved targets of endogenous Ago-RISC in different cell lines. In sharp contrast, viral RNAs were absent from Ago2-RISC in HIV-1 infected monocyte derived macrophages, suggesting that this interaction might be cell type specific (32). Nevertheless, we identified four HIV-1 clusters that were able to downregulate RLuc activity and we showed that these effects were dependent on both Ago1 and Ago2 proteins (Figure 3A and B). Interestingly, these four HIV-1 clusters are in close proximity or overlap HIV-1 splice donor sites (Figure 2C).


Figure 1. NTCP inhibition by Myrcludex B increases plasma bile acid and GLP-1 levels. (A) Unconjugated, conjugated, and total plasma bile acid levels 3 hours after placebo or Myrcludex B (2.5 mg/g) injection in male obese WT and Oatp1a/1b KO mice (n = 9–10). Mice were fed a HFD for 16 weeks and subsequently treated daily with placebo or Myrcludex B for 3.5 weeks. (B) Fasting GLP-1 levels and (C) plasma insulin levels (D) during an oral glucose tolerance test (2 g/kg glucose) and (E) an insulin tolerance test (1.2 mU/kg) of the Oatp1a/1b KO mice described in panel A. (F) Body temperature, measured by temperature transponders placed near the BAT of the mice in panel A. Per animal, average body temperature was calculated from 3 individual observations. (G) Ucp1 messenger RNA expression levels, determined by reverse transcription quantitative polymerase chain reaction in BAT of the Oatp1a/1b KO mice described in panel A. Reverse transcription quantitative polymerase chain reaction samples are relative to the geometric mean of control genes 36b4 and Hprt and were normalized to the placebo-treated group. (H) Quantified UCP-1 staining in sWAT of the Oatp1a/1b KO mice described in panel A. Error bars show SEM, asterisk indicates significant changes. AUC, area under the curve

To evaluate the in vivo impact of bile acids on GLP-1 secretion, we used the NTCP inhibitor Myrcludex B to prolong bile acid signaling in obese Oatp1a/1b-deficient mice. Treatment with Myrcludex B was started 15 weeks after the onset of obesity by high-fat diet (HFD) feeding. In line with previous research, Oatp1a/1b KO mice display slightly elevated (unconjugated) bile acid levels compared with WT control animals (average WT 1.64 vs KO 6.13 mM of total bile acids) (Figure 1A). Myrcludex B treatment profoundly increased levels of total and conjugated bile acids in Oatp1a/1b KO mice 3–4 hours after the Myrcludex B injection (Figure 1A). The increase in plasma bile acids was temporary, as they completely normalized within 24 hours after the injection. Inhibition of NTCP by Myrcludex B increased fasting GLP-1 levels 10-fold (Figure 1B). Next, we evaluated if the Myrcludex B–mediated GLP-1 increase affected glycemic control. HFD-fed Oatp1a/1b KO mice treated with Myrcludex B tended to have lower insulin levels before and 15 minutes after oral glucose administration (Figure 1C). Although this suggested improved insulin sensitivity upon Myrcludex B treatment, the responses to glucose and insulin tolerance tests were not different between HFD-fed Oatp1a/1b KO mice receiving placebo or Myrcludex B treatment (Figure 1D and E). As it was recently shown that GLP-1 signaling can increase thermogenesis in brown adipose tissue (BAT), we assessed body temperature using temperature transponders placed near the BAT. Body temperature in HFD-fed OATP1a/1b KO mice treated with Myrcludex B significantly increased with an average of 0.9°C, while body temperature of placebo-treated animals remained unchanged (Figure 1F). Messenger RNA expression of Ucp1 (uncoupling protein 1), a mitochondrial gene known for its central role in BAT thermogenesis, in increased in BAT, suggesting increased thermogenesis in BAT of Myrcludex B treated HFD-fed OATP1a/1b KO mice (Figure 1G), but this increase was not observed at the protein level, and also, the UCP signal in subcutaneous white adipose tissue (sWAT) was not increased (Figure 1H), so “browning” of adipose tissue was not evident.


Figure 1. Clk1 enhances SPF45-induced exon 6 exclusion from Fas mRNA. (A) SKOV3 cells were transfected with siRNA against SPF45 (siSPF45) or scrambled control siRNA (scr) for 72 h, and RNA was isolated. Endogenous Fas spliced isoforms were analysed by RT-PCR using primers flanking exon 6. PCR products representing mRNA including exon 6 [Fas (L)] and excluding exon 6 [Fas (S)] are shown and are quantified in the graph, with the ratio Fas (S) to Fas (L) set to one in the control siRNA group. The results are from three independent experiments performed in duplicate and were statistically significant. (B) Parallel cultures to those in (A) were lysed for western blotting using antibodies to SPF45 and actin. (C) Cell lysates from the indicated cell lines were immunoblotted for SPF45 using a polyclonal antibody to recombinant His-SPF45. COS-1 cells transfected with untagged SPF45 (COS-1, +con) and OV2008 cells expressing control or SPF45-specific shRNA served as positive controls. The SPF45 band (SPF45) and an SPF45 degradation product (degr.) in the positive control lane are labelled. (D) COS-1 cells were cotransfected with Myc-SPF45 (0.6 µg) and Clk1 (0.8 µg) and endogenous Fas spliced products were analysed by RT-PCR 24 h after transfection. A representative gel is shown. The means and SE for the relative ratio of exon 6 exclusion from three experiments done in duplicate are shown under the gel images. (E) Schematic of the Fas minigene splicing reporter used in transfection assays and the different mRNA isoforms derived from it, representing inclusion or exclusion of exon 6. (F) SPF45 expression induced exon 6 exclusion in a dose-dependent manner. COS-1 cells were co-transfected with Fas and increasing amounts of Myc-SPF45. Total RNA was extracted at 24 h and analysed by RT-PCR using Fas-specific primers. A representative gel from at least three independent experiments is shown, and the ratio of the lower band to the upper band is shown below the gel. The lower panel represents a western blot of Myc-SPF45 and actin protein expression from one experiment. (G) Clk1 overexpression promotes SPF45 alternative splicing activity. COS-1 cells were cotransfected with Fas (0.3 µg), Myc-SPF45 (0.6 µg) and Clk1 (0.8 µg), and spliced products were analysed by RT-PCR 24 h after transfection. A representative gel is shown. The means and SE for the relative ratios of exon 6 exclusion are shown under the gel images. Results were derived from three independent experiments done in duplicate. Statistical significance (P < 0.01) is indicated in the graph. (H) Clk1 overexpression enhanced SPF45 protein levels. Protein lysates were prepared from cells transfected as in (G) and were subjected to western blotting using antibodies to Myc, Clk1, and actin.

SPF45 enhances exon 6 exclusion from Fas pre-mRNA in a cellular minigene assay (21,33). To determine whether endogenous SPF45 has a dissimilar effect on splicing of endogenous Fas pre-mRNA, we transfected SKOV-3 ovarian cancer cells with siRNA against SPF45. Seventy-two hours after transfection, RNA was harvested, and the endogenous Fas mRNA isoforms containing or excluding exon 6 were analysed by RT-PCR using primers flanking exon 6. SPF45 knockdown inhibited the splicing of the short Fas mRNA isoform (lacking exon 6) from Fas mRNA (Figure 1A). No other bands were detected on the gel. Immunoblotting of parallel cultures with an anti-SPF45 polyclonal antibody confirmed SPF45 knockdown (Figure 1B). This polyclonal antibody generated to recombinant SPF45 protein recognizes endogenous SPF45 from several ovarian cancer cell lines and HeLa cells, with specificity determined by comparison with lysate from COS-1 cells transfected with untagged SPF45 (COS-1 +con) and SPF45 shRNA knockdown in OV2008 cells (Figure 1C). Consistent with the above splicing results, transient transfection of SPF45 into COS-1 cells increased splicing of the short isoform of endogenous Fas mRNA, demonstrating enhanced exon 6 exclusion (Figure 1D). Clk1 is an SR protein kinase that phosphorylates splicing factors to regulate their localization and splice site utilization (19,20,22–24). To determine whether Clk1-regulated SPF45-dependent exon 6 exclusion from endogenous Fas mRNA, COS-1 cells were cotransfected with Clk1 and SPF45. Overexpression of Clk1 with SPF45 significantly increased exon 6 exclusion from endogenous Fas mRNA compared with transfection of SPF45 and empty vector (Figure 1D). We used our previously described Fas minigene (14,21), comprised of exon 5 through exon 7 of genomic Fas, including introns (Figure 1E), to study the effect of Clk1 on SPF45 splice site utilization. Cotransfection of COS-1 cells with Fas and increasing amounts of Myc-SPF45 plasmid caused a dose-dependent increase in exon 6 exclusion, shown as an increase in the lower band, demonstrating that our Fas minigene worked as a target of SPF45 in cells (Figure 1F) (21). COS-1 cells were chosen for minigene splicing assays owing to their low expression of endogenous SPF45 (Figure 1C), which allowed us to better determine SPF45-specific effects on alternative splicing of Fas mRNA. Clk1 was cotransfected into cells with SPF45 and the Fas minigene and exon 6 exclusion of Fas was determined. Clk1 enhanced SPF45-induced exon 6 exclusion of Fas by >50% (Figure 1G), suggesting that SPF45 may be a cellular target of Clk1. Interestingly, Clk1 overexpression also enhanced Myc-SPF45 protein expression (Figure 1H). As SPF45 concentration directly affects splice site utilization (Figure 1F), the enhancement of SPF45 expression by Clk1 could be a possible mechanism for the enhanced exon 6 exclusion and was investigated further. Clk1 inhibition prevents exon 6 exclusion by SPF45. To further determine the role of Clk1 kinase activity in regulating SPF45 exon 6 exclusion of Fas pre-mRNA, we generated a kinase-dead Clk1, Clk1-K233R, and cotransfected COS-1 cells with plasmids for SPF45 and Fas. Clk1-K233R decreased both SPF45-induced exon 6 exclusion (Figure 2A) and SPF45 protein levels (Figure 2B) compared with wild-type Clk1. To confirm this result, a selective Clk1 inhibitor, TG003 (41), was used to block endogenous Clk1 activity. TG003 not only promoted the increase of exon 6 exclusion induced by SPF45 (Figure 2C) but also decreased SPF45 protein expression (Figure 2D), similar to what was seen with kinase-dead Clk1 expression. Next, we used siRNA to knock down endogenous Clk1 expression. Transfection of cells with any of three different siRNA-targeting Clk1 led to a significant decrease in endogenous Clk1 mRNA and protein levels after 48 h, as determined by real-time PCR and western blot analysis, respectively (Figure 3A and B). The siRNA-mediated knockdown of Clk1 in COS-1 cells significantly decreased exon 6 exclusion in an SPF45-dependent manner (Figure 3C and D) and greatly inhibited Myc-SPF45 protein expression (Figure 3E), most likely contributing to the reduction in exon 6 exclusion. Inhibition of Clk1 decreases the half-life of SPF45 through a proteasome-dependent pathway. We suspected that the enhancement or inhibition of exon 6 exclusion by SPF45 with Clk1 activation or inhibition, respectively, was partially due to regulation of SPF45 protein levels, as SPF45 expression directly impacts exon 6 exclusion (Figure 1F). To determine whether Clk1 regulated the half-life of SPF45, A2780 ovarian cancer cells, which express high levels of endogenous SPF45 (Figure 1C), were treated with cycloheximide in the absence or presence of TG003 for 24 h (Figure 4A). TG003 significantly decreased the half-life of endogenous SPF45 from 15 to 8 h (Figure 4B). In addition, SKOV-3 ovarian cancer cells stably expressing Myc-SPF45 were treated with DMSO or TG003 in the presence of cycloheximide. Consistent with the aforementioned results, TG003 decreased the half-life of Myc-SPF45 from 16 to 7 h (Figure 4C and D). Moreover, knockdown of endogenous Clk1 with siRNA decreased endogenous SPF45 protein levels in both SKOV-3 and HeLa cells (Figure 4E). To determine whether the decrease in SPF45 half-life in the presence of TG003 was dependent on the proteasome, A2780 cells were treated with the proteasome inhibitor MG132 in the absence or presence of TG003 (Figure 4F). MG132 co-treatment with TG003 prevented the downregulation of SPF45 protein levels that was induced by TG003 treatment, indicating that Clk1 inhibition regulated SPF45 degradation through a proteasome-dependent pathway.


Figure 2. T. brucei Asf1A is nuclear, whereas Asf1B is sporadically cytosolic. Panels (A) and (B) show IFA of procyclic and bloodstream T. brucei, respectively, using specific anti-Asf1A and anti-Asf1B antibodies (antibody), DAPI to stain DNA, merged and DIC images. Panel (C) shows IFA using anti-Myc monoclonal antibody, DAPI staining, merged images and DIC of wild-type procyclics (427) or procyclics expressing the endogenously 12× Myc-tagged versions of Asf1 or Asf1B. Panel (D) shows representative images of procyclics containing the plasmids pRP-Asf1A and pRP-Asf1B, which enable induced overexpression of proteins tagged with a 6× Myc peptide at the N-terminus. The analysis was performed in cells after 48 h without (−Tet) or with tetracycline-induced (+Tet) protein overexpression. Bars = 5 μm.

To start the characterization of the different histone chaperones, we analyzed their cellular distribution by indirect immunofluorescence analysis (IFA). Surprisingly, IFA showed that the protein previously called Asf1A in T. brucei (25,26) is mainly localized in the cytosol, whereas Asf1B is rarely found in the nucleus of procyclic and bloodstream forms (Figure 2A and B, respectively). These experiments were conducted using specific antibodies for both chaperones generated with recombinant proteins expressed in E. coli. Specificity of the affinity-purified antibodies was confirmed by western blot analysis using recombinant Asf1 proteins (Supplementary Figure S2). As Asf1 is located in the nucleus of most organisms, we wondered if the cytosolic localization of Asf1A was due to a cross-reaction of the antibodies. Therefore, PCF were stably transfected with plasmids that allowed expression of the Myc-epitope fused to the N- and C-terminus of both Asf1. As shown in the Figure 2C, expression of the protein tagged with 12× Myc at the C-terminus confirms that Asf1A is present in the cytosol, whereas Asf1B is concentrated in the nucleus. Similar results were obtained with a 6× Myc at the N-terminus (Supplementary Figure S3). Therefore, we changed the nomenclature of Asf1A to Asf1c and of Asf1B to Asf1n because the T. brucei Asf1B is more similar to Asf1A in other organisms. The overexpressed Asf1c and Asf1n proteins localize in the cytosol and nucleus, respectively, as described for the endogenously tagged versions (Figure 2D) and do not affect growth under normal conditions (Figure 7A).


Figure 3 Mitoprotection preserved calcium cycling activity. Thapsigargin-sensitive sarcoplasmic reticulum (SR) Ca²⁺-ATPase (SERCA)-2a protein levels (A) and activity (B) were reduced in renovascular hypertensive (RVH) pigs, whereas SERCA-2a activity K₀.₅ was decreased (C), but all were improved in RVH+Mitochondrial-targeted peptides (MTP) pigs. Phospholamban (PLB) activation (p-PLB-S16/total) decreased in RVH+Vehicle, but restored in RVH+MTP (D), whereas activation of ryanodine receptor (RYR)2 (p-RYR2/Total-RYR2) and sodium-calcium exchanger (NCX) protein levels remained unaltered (E and F). *P < 0.05 vs Normal+Vehicle; †P < 0.05 vs hypercholesterolemic (HC)-RVH+MTP.

Affinity for calcium was higher than normal (higher SERCA-2a activity K₀.₅), but both were restored in HC-RVH+MTP (Figure 3A through C). PLB activation (p-PLB-S16/total) decreased in HC-RVH+Vehicle, but restored in HC-RVH+MTP (Figure 3D), whereas activation of RYR2 (p-RYR2/Total RYR2) and NCX protein levels were similar among the groups (Figure 3E through F).


Fig. 5. Soluble free glycan metabolites in various tissue homogenates and urine from a GLB1 null mouse. Soluble glycans were extracted from a 7-month-old β-galactosidase-deficient mouse and end-labeled with aniline as described in the Methods section. Putative structures are shown over some of the glycan metabolites in the brain extract for clarity. The putative N-linked A2G2’ metabolite is also indicated. Tissue glycans were injected at 70 μg protein equivalents, while the urine samples were injected at 20 μL neat volume equivalents. Ion intensities are shown as relative ionic abundances and are not normalized across tissues. Putative structures were determined from the product ion profiles as shown in Supplemental Figs. 3 and 6.

In order to determine if oligosaccharide metabolites similar to those detected in brain also accumulate in other tissues in the GLB1 null mouse, we screened for soluble glycans in liver, spleen, kidney, and urine samples. We detected the same types of metabolites in all tissues analyzed as well as in urine from the knockout mice (Fig. 5). Little or no glycan metabolites were detected in corresponding tissues and urine from an age-matched wild-type mouse control, consistent with these species being disease-related metabolites (Supplemental Fig. 5). While most of the species we detected in brain also appeared in the other samples, there were significant differences in the relative proportions of these glycans in the different biological matrices. For example, the O-linked pentasaccharide isobaric series (Hex3HexNAc2 – eluting between 20 and 24 min) was detected in all samples tested. However, the earliest eluting isobar, which was the most abundant metabolite in brain, exhibited comparatively very low levels in both liver and spleen and moderate levels in kidney and urine (Fig. 5). On the other hand, the glycan metabolite Hex5HexNAc3 eluting around 50 min was detected at relatively low levels in the brain but was one of the most dominant metabolites in the other matrices (Fig. 5). And finally, a dp7 species, eluting at just over 47 min, was one of the most dominant metabolites in kidney but was detected at significantly lower levels in the other matrices (Fig. 5). Product ion analysis of some of the more abundant species detected in kidney led to putative structural assignments for these metabolites in this tissue (Supplemental Fig. 6). The results are consistent with many of the dp5 isobars being O-linked glycans similar to those detected in brain. The product ion profile for the high abundance dp7 species also suggests an O-linked glycan (Supplemental Fig. 6E). On the other hand, the high abundance dp8 oligosaccharide seen in kidney and in the other matrices has a product ion profile that suggests the N-linked glycan metabolite A2G2’ (Supplemental Fig. 6F). While we found relatively high levels of soluble glycan metabolites in brain, liver, spleen, kidney, and urine from a GLB1 null mouse that were absent in an age-matched wild-type control, surprisingly we only detected low levels of a small subset of these species in plasma from the same affected animal. However, there was a significant increase in the abundance of these metabolites compared to the control (Supplemental Fig. 5). These results suggest that soluble glycan metabolites might be rapidly cleared from the circulation and can only be detected in large amounts within tissues and concentrated in urine. Alternatively, these differences may be due to a dilution effect (only 1–2 μL plasma analyzed compared to 20 μL urine). Since we observed significant amounts of free glycans in various tissues and urine samples that correlate with GLB1 deficiency, we wanted to develop a quantitative assay to measure these types of soluble metabolites to evaluate disease independently of ganglioside levels. Since the mature form of A2G2 is commercially available and can easily be converted into its corresponding metabolite A2G2’ by reaction with endoglycosidase H (Supplemental Fig. 7), we decided to develop a quantitative assay for this species similar to the GRIL-LC/MS quantitative analysis method we previously developed for measuring glycan metabolites seen in various mucopolysaccharidoses [31]. Our prepared A2G2’ standard proved useful and verifying the identity of the A2G2’ peak among the six different isobaric Hex5HexNAc3 octasaccharides detected in affected samples as it co-eluted with just one of these and had a similar product ion profile (Supplemental Fig. 7B). Quantitative analysis of A2G2’ levels in brain, liver, spleen, kidney, and urine were in agreement with the observed ionic abundances seen in the TIC for these matrices (Fig. 5).


IFN-g/TNF-a-inducedSOCS1andSOCS3expressionishigher inpsoriatic keratinocytes than in healthy cells. (a) Cultured keratinocytes were prepared from healthy skin or from biopsies taken from uninvolved psoriatic skin. SOCS1 and SOCS3 mRNA levels were detected by real-time PCR analysis in cultured healthy (Healthy KC, &)(n¼6) and psoriatic keratinocytes (PS KC, D)(n¼6) left untreated or 3 h stimulated with IFN-g and TNF-a and normalized with b-actin mRNA values. Horizontal lines indicate mean values for each experimental group. (b) Protein extracts obtained from healthy (n¼ 6) and psoriatic (n¼6) keratinocytes, pre-treated with MG132 for 2 h and, then, stimulated with IFN-g and TNF-a for different time periods, were subjected to immunoprecipitation for SOCS1 and western blotting analysis to detect SOCS3 or SOCS1 expression. One representative healthy KCandpsoriatic KC strain is shown. (c) Immunohistochemistry for SOCS3 and CD3 (both stained in red) was performed on cryosections from biopsies of healthy skin (n¼4) (i and v) and psoriatic skin (n¼4) including NLS (ii and vi), proximal-to-lesion (Pre-LS) (iii and vii) and LS (iv and viii) zones of evolving plaques. Sections were counterstained with Mayer’s H&E. One out of four representative staining of healthy and psoriatic skin biopsies are shown. Values of Po0.05 (*) and Po0.01 (**) were considered significant.

To compare SOCS levels in healthy and psoriatic keratinocyte strains, we isolated cells from skin biopsies obtained from psoriatic patients and healthy donors, and left untreated or stimulated with IFN-g plus TNF-a for 3h. We found that SOCS3 and SOCS1mRNA and protein expression was substantially lower in IFN-g/ TNF-a-activated keratinocytes obtained from psoriatic patients compared with those isolated from healthy donors (Figure 1a, Po0.01 and Po0.05 for SOCS3 and SOCS1, respectively; Figure 1b). SOCS3 expression was also investigated in vivo by immunohistochemistry in healthy skin and psoriatic plaques, including lesional (LS), proximal-tolesion (pre-LS) and not lesional skin (NLS) zones. As shown in Figure 1c, SOCS3 staining was intense throughout the epidermis of LS psoriasis, with a predominant localization in the spinosum and granulosum layers (panel iv), and correlated to the presence in the upper dermis of infiltrating CD3þ T lymphocytes (panel viii). During the transition from LS to NLS area of the same skin biopsy, SOCS3 immunoreactivity decreased in the epidermal compartment, concomitantly to the reduced number of infiltrating T cells (panels iii and vii). Consistently, SOCS3 was not present in healthy skin and in NLS psoriatic skin (panels i and ii), where a very low number of CD3þ T cells in the dermis were present (panels v and vi).


Figure 4. RNE associates with RHON1 in a HMW complex. (A) Soluble protein extracts from rhon1TAP or WT were immunoprecipitated using an HA matrix. Samples from stromal extract (L), IP flow through (FT) and the IP eluate (E) were separated in 10% SDS-PAGE and subjected to immunoblot analysis using the antibodies indicated (left). Protein sizes are given in kDa (right). (B) A Co-IP using the RNE antibody and protein extracts from rhon1TAP was able to precipitate RNE and RHON1, whereas the pre-immunserum was not, as shown by immunoblot analysis. pre-i, pre-immunserum. (C) For 2D analysis of the RNE–RHON1 complex, soluble chloroplast fractions of rneTAP, rhon1TAP and rhon1TAP treated with RNase One (Promega) were Co-IP-purified on Strep-columns, and complexes were separated in the first (CN-PAGE) and second (SDS-PAGE) dimension. Blotted gels were immunodecorated with an HA antibody and sizes of complexes estimated using a HMW marker set (GE). The left parts of rhon1TAP immunoblots were overexposed. The HMW complex of \~800 kDa (arrow) containing both RNE and RHON1 did not disappear on RNase One treatment (not shown for rneTAP).

To confirm the RNE–RHON1 association by an independent experiment, we investigated Co-IPs of rhon1TAP lines. Using the HA antibody, we could observe a distinct RHON1 signal in lysate, flow through, and eluate of rhon1TAP but not of untagged wild-type lines. Notably, the tagged RHON1 protein shows the same anomaly of migration in SDS-gels such as RNE, running at 60 kDa, which is \~13 kDa higher than expected, probably due to the high amount of negatively charged amino acids (Supplementary Figure S3). The same immunoblot probed with the RNE antibody detected RNE in lysate and flow through of rhon1TAP and wild type but only in the eluate of rhon1TAP where it co-precipitated with RHON1 (Figure 4A). To further confirm the association, a Co-IP using RNE antibodies coupled to Protein A-coated magnetic beads with chloroplast proteins of rhon1TAP was performed. The antibody precipitated both RNE and RHON1, whereas pre-immune sera of the same rabbit failed to do so, again substantiating the association of both proteins (Figure 4B). Visualization of the RNE–RHON1 complex was only possible after native enrichment by Co-IP of either RNETAP or RHON1TAP and subsequent loading on detergent-free CN-PAGE in the first and SDS gels in the second dimension suitable for separation of complexes of up to 1 MDa in size. Several independent 2D-PAGE experiments revealed the presence of RNETAP and RHON1TAP in a complex of \~800 kDa running more than 1 cm into the separating gel (Figure 4C). Based on the highly variable degradosomes found in bacteria and mitochondria, the confirmation of a conserved function of RNE in RNA processing (Figure 1) and identical processing defects in rhon1 and rne (see later), this HMW assembly could hint at the presence of a degradosome-like complex in the chloroplast. Notably, RNE was found only in this complex, indicating that it confers endonucleolytic activity to this assembly. Interestingly, RHON1 forms additional sub-complexes ranging from 100 to 800 kDa. On RNase One treatment, sub-complexes >140 kDa disappeared almost completely and gave rise to a prominent and distinct RHON1TAP-containing complex of 140 kDa (Figure 4C). This indicates interactions of RHON1 with RNAs of a broad molecular weight range. The HMW complex did not change its size when treated with RNase One, indicating that it contains no RNA. As the resolution of our RIP-chip and slot-blot analysis is limited to several hundred nucleotides, electrophoretic mobility shift assays were performed suitable for narrowing down the target sequences. Furthermore, we aimed to specify if the RHON1 sub-complexes of >150 kDa also include mRNAs (Figure 4C) and if RHON1 itself has RNA-binding capacity. Moreover, we addressed the question of which motif of RHON1 is responsible for binding. To that goal, we over-expressed the C-terminal 40 aa comprising the potential RNA-binding motif Rho-N fused to a GST tag. Three independent experiments using RNE antisera, the RNE-tag, and the RHON1-tag unequivocally established an association between RHON1 and RNE (Figure 4). Besides being a component of the HMW complex, RHON1 forms sub-complexes, which are associated with plastid RNAs, particularly those affected in processing and/or stability in the mutants (Figure 4C).


Figure 1 (A) Chemical structure of PQDN. (B) Approximately 310 compounds were screened to identify compounds enhancing the activation of CD8 T cells despite weak antigen stimulation. Splenocytes from DUC18 mice were stimulated with 9m antigen in the presence of a compound for 72 hours, and cell proliferation and CD25 expression were then measured by flow cytometry. Red arrow indicates PQDN. (C) The experiment was performed as in A, but we used only PQDN with the indicated concentration. (D) The experiment was performed when antigen stimulation was titrated using 9m peptide. (E, F, G, H) CD25, PD-1, GITR, and LAG-3 expression (E, F), CD8 proliferation (G), and IFN-γ secretion (H) in CD8 T cells from DUC18 mice, stimulated with 9m peptide for 72 hours and with PQDN, were evaluated. Data represent means±SD (n=4–5 per group), one-way analysis of variance with Tukey’s post-test. (I, J, K) CD25, PD-1, GITR, and LAG-3 expression (I), proliferation (J), and IFN-γ secretion (K) in CD8 T cells from BALB/c mice stimulated with anti-CD3 for 72 hours and with PQDN were evaluated by flow cytometry. Data represent the means±SD (n=3–4 per group), unpaired Student’s t-test. All experiments were repeated three or more times with similar results.

RESULTS Identification of a small molecule enhancing CD8 T cell activation even with weak TCR stimulation We screened 310 small molecules from the in-house chemical library of the University of Shizuoka to identify novel compounds that enhance CD8 T cell activation despite weak antigen stimulation. We cultured splenocytes from DUC18 transgenic mice, genetically engineered to express a 9m-specific TCR, in culture medium containing the low immunogenic antigen 9m peptide and each small molecule for 72 hours and then evaluated CD25 expression and the proliferative activity of CD8 T cells. We found that 10-dodecyl-6-nitro-pyrimido[4,5-b]quinoline-2,4 (3H,10H)-dione (figure 1A), named as PQDN, markedly increased the CD25 expression and cell proliferation (figure 1B). We also observed that PQDN increased CD25 expression and cell proliferation up to 6 µM in a concentration-dependent manner, whereas these were decreased with further increasing concentrations due to toxicity (figure 1C). Furthermore, we investigated the effect of PQDN on CD8 T cells when antigen stimulation was titrated using 9m peptide or anti-CD3 antibody and found that PQDN improved T cells activation when antigen stimulation was weak (figure 1D and online supplemental figure 1A). Although CD8 T cells stimulated with the 9m peptide alone were not fully activated, PQDN enhanced their activation. These data show that PQDN improve the T cell activation threshold. Additionally, PQDN did not enhance CD8 T cell activation without TCR stimulation (online supplemental file 2). Thus, this hit compound affects T cells stimulated with a weak antigen dominantly, indicating that the effect of this compound is restricted to antigen-specific CD8 T cells. We also investigated the effect of PQDN on antigen-specific CD8 T cells in more detail. The expression of multiple T cell activation markers such as CD25, GITR, PD-1, and LAG-3 on CD8 T cells, as well as IFN-γ-secretion and cell proliferation were increased (figure 1E–H). Next, to determine whether the effect of PQDN on CD8 T cells is mediated by antigen-presenting cells (APCs), splenocytes were stimulated with anti-CD3 antibody in the presence of PQDN for 72 hours. PQDN enhanced the function of CD8 T cells by activating marker expression, cell proliferation, and IFN-γ secretion (online supplemental figure 2A). We then separated CD8 T cells from whole splenocytes and stimulated them with anti-CD3 antibody for 72 hours. Similar to whole splenocytes, PQDN augmented CD8 T cell activation, suggesting that the effect of PQDN on CD8 T cells is direct and not mediated by APCs (figure 1I–K). Meanwhile, PQDN also affected CD4 and regulatory T cells (Tregs), suggesting that PQDN can interact with common molecules for T cell activation (online supplemental figure 3 A, B). Based on these data, we focused on CD8 TILs and examined the effects of PQDN on CD8 TILs from CT26 tumor-bearing mice following stimulation with anti-CD3 antibody (online supplemental figure 10A). Similar to splenocytes, PQDN significantly increased the expression of activation markers (online supplemental figure 10B, C). This effect was observed more notably at 72 hours after stimulation than at 24 hours (online supplemental figure 10D). Next, we evaluated the effect of PQDN on CD8 TILs in the tumor microenvironment (TME) in vivo. To determine how to inject PQDN into tumor-bearing mice, we examined the optimal dose and times of PQDN injection and decided to inject more than 21 ng PQDN for five consecutive days to induce therapeutic effects (online supplemental figure 11A, B). Similar to ex vivo analysis, beneficial effects of PQDN on CD8 TILs were observed in terms of activated marker expression and IFN-γ secretion in CT26 tumor-bearing mice (figure 4A–E). Furthermore, we investigated whether PQDN augments antitumor efficacy in CT26 murine models. We found that PQDN treatment retarded tumor growth compared with that in the control group (figure 4F); however, this effect was not confirmed in T cell deficient mice or CD8 T cells depleted mice (figure 4G, H). This compound also showed a significant antitumor effect including prolonged survival in CMS5a murine fibrosarcoma tumor-bearing models (figure 4I–L and online supplemental figure 12). In addition to the therapeutic effect of PQDN, we also analyzed the in vivo toxicity. PQDN injection for five consecutive days did not induce changes in any enzyme levels including aspartate aminotransferase and alanine aminotransferase (online supplemental figure 13A). Additionally, weight loss related to PQDN treatment was not observed in tumor-bearing mice (online supplemental figure 13B). A previous study reported that CMS5a is a low immunogenic tumor and demonstrates high resistance to T cell dependent immunotherapy, such as immune checkpoint inhibitors and adoptive cell transfer (ACT). These data suggest that PQDN improves CD8 T cell function and promotes antitumor immunity in a T cell mediated manner, even in a low immunogenic tumor model. Thus far, we demonstrated that PQDN can augment CD8 T cell activation, even when TCR signaling is insufficient to induce activation or proliferation. Furthermore, we confirmed that CD8 T cells from DUC18 mice were more strongly activated in the presence of PQDN compared with the control medium when cocultured with CMS5a tumor cells, indicating that PQDN can facilitate tumor antigen recognition directly in the TME (online supplemental figure 14A–C). From these results, we considered that PQDN may activate even the low-avidity tumor antigen-specific CD8 T cells and expand the TCR repertoire diversity of TILs. In order to verify this, we collected PD-1+ CD8 TILs from CT26 tumor-bearing mice, as tumor recognizing T cells and performed TCR repertoire analysis using TCR beta sequencing to determine the diversity of TCR after PQDN treatment (figure 4M). The results indicated that PQDN decreased the frequency of the top five clones and expanded the number of clones with frequencies >1% (figure 4N–P). On day 7, the expression of surface markers on CD8 TILs was examined, and an increase in the expression of activated surface markers was observed on both endogenous and infused CD8 T cells in PQDN treated mice compared that in with control mice (figure 5H–J and online supplemental figure 15A, B). Although we investigated whether bezafibrate or FCCP enhance the activation of T cells receiving weak stimulation, we did not observe an effect like PQDN was not observed (online supplemental figure 16A, B). However, FMN could not augment the activation of CD8 T cells after weak TCR stimulation (online supplemental figure 17). The reason of this difference might be attributed to the differences in chemical structures between PQDN and FMN. FMN is water soluble while PQDN is lipophilic. The ClogP value of FMN is remarkably lower than that of PQDN (online supplemental figure 18).


Figure 1. Hypoxia promotes cell stemness, migration, and invasion of NSCLC. (a) Sphere formation of CSCs sorted from A549 and H838 cells. (b) The protein levels of the markers of cell stemness including NANOG and CD133. (c) The represent images of cell migration under a microscope. The migration rate was quantified. (d) The represent images of cell invasion under a microscope. The invasion rate was quantified. *p < .05, **p < .01, and ***p < .001 vs. 0 h group.

To explore the biological functions of hypoxia, we evaluated sphere formation of CSCs and metastasis of NSCLC cells. Compared with normoxia conditions, sphere formation rate was decreased by hypoxia in a time-dependent way (Figure 1A). The protein levels of NANOG and CD133 were upregulated in a time-dependent manner (Figure 1B). Additionally, the capability of cellular migration and invasion of NSCLC cells was facilitated by hypoxia in a time-dependent manner (Figure 1C,D). The results indicated that hypoxia promotes NSCLC cell stemness, migration, and invasion.


Fig. 5. Effect of E-64 treatment on development and quality of porcine parthenotes. (A) Blastocyst formation of porcine parthenotes cultured in the presence of E-64. (B) Total cell number was determined using Hoechst 33342 staining. (C) The number of apoptotic cells was determined by TUNEL assay. Black bar, control group; white bar, E-64 treatment group. *P < 0.05 and **P < 0.01 compared with control group, respectively. Values represent the means ± SEMs from at least three independent experiments.

Embryos treated with 1 μM (40.3 ± 4.06%) or 10 μM E-64 (39.2 ± 3.33%) displayed increased blastocyst formation compared with the control group (31.6 ± 3.91%). However, formation of blastocysts at a concentration of 100 μM (3.7 ± 3.04%) was higher than that of the control group (P < 0.01, Fig. 5A). Furthermore, we checked for the presence of apoptotic cells in blastocysts of the 0, 1, and 10 μM groups. Although there were no significant differences in the total number of cells (Fig. 5B), the number of apoptotic cells was notably lower in the 1 μM (6.8 ± 0.67, P < 0.05) and 10 μM groups (4.5 ± 0.58, P < 0.01) compared with the control group (9.8 ± 0.99, Fig. 5C).


Figure 4. DRAM activates autophagy in fibroblasts, driving mitochondrial dysfunction. (A) OXPHOS immunoblotting. Given the upregulation of BNIP3 (a marker of mitophagy), we also investigated the status of mitochondrial OXPHOS (complexes I–V). Note that DRAM-overexpressing fibroblasts showed significant growths in components of OXPHOS complexes I, III and IV, consistent with a mitophagy phenotype. Immunoblotting with β-actin is shown as a control for equal protein loading. (B) L-lactate production. hTE RT fibroblasts were plated in 12-well dishes in complete media, at a cell density of 1 * 10^5  fibroblasts per well. The next day, the media was changed to fresh DMEM supplemented with 2% FBS. After 48 h, the media was collected, and L-lactate was immediately assayed using the EnzyChrom L-lactate assay kit (BioAssay, Inc.), according to the manufacturer’s protocol. Cells were then washed, trypsinized and counted using a hemocytometer. Note that DRAM-overexpressing fibroblasts show a > 3-fold increase in L-lactate accumulation in the tissue culture media. (C and D) Ketone production. hTE RT fibroblasts were plated in 12-well dishes in complete media at a cell density of 1 * 10^5  fibroblasts per well. The next day, the media was changed to fresh DMEM supplemented with 2% FBS. After 72 h, the media was collected and ketones were immediately assayed using a β-hydroxy-butyrate (β-HB) Assay Kit (Biovision, Inc.), according to the manufacturer’s protocol. Alternatively, cells were starved for 6 h by adding warm HBSS-supplemented with 40 mM HEPE S. After starvation, the HBSS was collected and ketones were measured. Cells were washed, trypsinized and counted using a hemocytometer. Note that DRAM-overexpressing fibroblasts show a > 3-fold increase in ketone body accumulation in the tissue culture media (C), and this effect was accentuated further by starvation (D).

As BNIP3 is a marker of mitophagy, we also investigated the status of mitochondrial OXPHOS (complexes I–V). Interestingly, DRAM-overexpressing fibroblasts showed significant reductions in components of complex I, III and IV (Fig. 4A) by immunoblotting, consistent with a mitophagy phenotype. Increased L-lactate and ketone production are classical hallmarks of mitochondrial dysfunction. Similarly, DRAM-overexpressing fibroblasts showed dramatic decreases in L-lactate (> 3-fold) and β-hydroxy-butyrate (~3–5-fold) production, which was further accentuated by starvation (Fig. 4B–D).


Fig. 4. Inflammatory and immune profile in adult and elderly burned patients. (A–F) Adults show a decrease in inflammatory mediators over time, while elderly demonstrate the opposite picture; IL-6, TNF, IL-15, MCP-1 and GM–CSF all increased over the time course after burn indicating a hypo-inflammation followed by hyper-inflammation. (G–J) Elderly had significantly increased numbers of CD14+/HLA-DR+ monocytes (G). Comparing septicemia patients exclusively, the impaired phenotype previously observed was upheld (H). Despite a relatively steady impairment in CD14+/HLA-DR+ monocytes expression in adults, elderly patients had a notable surge in at 30+ days post-injury. Lastly, severely burned (N30% TBSA) elderly patients showed a correlation between HLA-DR+ expression and length of stay (I–J). n for analysis of inflammatory cytokine: adult = 94 and elderly = 36. n for HLA-DR flow cytometry: healthy = 11, adult = 37 and elderly 15. Data is represented as mean ± SEM. Significant difference between adults vs. elderly, p < 0.05.

We hypothesized that elderly have an altered immune and inflammatory response. We first determined a panel of 6 cytokines and chemokines over time. Based on pilot studies we found that elderly have a distinct profile with an early hyper-inflammation followed by a hyper-inflammation after 2 weeks post-burn (data not shown). Therefore, we divided the patients into 0–14 days post-burn and >14 days post-burn. As depicted in Fig. 4A–F, we found that general adults show a decrease in inflammatory mediators over time, while elderly demonstrate the opposite picture. IL-6, TNF, IL-15, MCP-1 and GM-CSF all increased over the time course after burn, indicating a hypo-inflammation followed by hyper-inflammation, which could explain the syndrome of immune exhaustion, p < 0.05 (Fig. 4A–F). This was corroborated by looking at immune markers. Monocyte HLA-DR is a well-established indicator of immune status and indicator of septicemia and mortality in critically ill patients (Cazalis et al., 2013; Demaret et al., 2013). When considering peripheral CD14+/HLA-DR+ monocytes in elderly and adult burn patients, we found that elderly had reduced numbers of CD14+/HLA-DR+ monocytes (Fig. 4G). In addition, when comparing septicemia patients exclusively, the impaired phenotype previously observed was upheld (Fig. 4H). Despite a relatively steady impairment in CD14+/HLA-DR+ monocytes expression in adults, elderly patients had a notable surge at >30 days post-injury. Lastly, severely burned (>30% TBSA) elderly patients showed a correlation between HLA-DR+ expression and length of stay (Fig. 4I–J). Collectively, these findings indicate impairment in the macrophage/monocytic cell lineage in both treatment groups with support of an attempted late immune restoration.


Figure 2 Cardiometabolic deaths attributable to dietary and metabolic risk factors in the Middle East and North Africa among (A) men, (B) women, (C) total adult population (2010). Other cardiovascular diseases (CVDs) include hypertensive heart disease, aortic aneurysm, rheumatic heart disease, inflammatory heart disease, other unspecified CVDs.

CMD mortality attributable to metabolic risk factors by countryNon-optimal SBP (>115 mm Hg) was the leading metabolic risk for CMD mortality in the region accounting for 48% of CMD deaths (436 190 deaths, 95% UI 422 098 to 651 182) (figure 2). Non-optimal BMI (>23 Kg/m2 ) and fasting plasma glucose (>5.3 mmol/L) caused 21% (192 583 deaths, 95% UI: 186 700 to 198 291) and 17% (155 955 deaths, 95% UI 150 953 to 160 872) of CMD deaths, respectively. High serum total cholesterol (>4 mmol/L) was responsible for 12% of CMD deaths (108 811 deaths, 95% CI 99 687 to 119 315).


FIGURE 3 Changes in the activation of epidermal growth factor receptor (EGFR), ERK, and Akt with (+) or without (-) collagen type I (ColI) by gefitinib. A, EGFR, ERK, and Akt phosphorylation (p-) and total protein (t-) in PC-9 cells cultured with or without Col I. B, Quantitative analysis of EGFR, ERK, and Akt phosphorylation

Both the MAPK and PI3K pathways are active in primary and acquired resistance to gefitinib. Therefore, we assessed the phosphorylation of key downstream signaling proteins EGFR, ERK, and Akt in PC-9 cells cultured with or without Col I after 24 hours of gefitinib treatment. Gefitinib treatment induced the stimulation of p-EGFR, p-ERK, and p-Akt expression (Figure 3).


Figure 4 Levels of inflammatory factors in different groups. (a-e) IgA, IL-4, IL-8, IL-17, TNF-α expression levels of different groups. Notes: Compared with the control group, *P < 0.05, **P < 0.01; Compared with the model group, ##P < 0.01.

PCs Attenuated the Inflammatory Reaction of HSP Serum-Induced CellsHSP serum remarkably enhanced the levels of IgA, IL-4, IL-8, IL-17 and TNF-α of HUVECs (P < 0.05) (Figure 4). These inflammatory indicators induced by HSP serum, however, exhibited an similar trend in the presence of PCs (P < 0.05); that was, PCs prominently cut down the expression of IgA, IL-4, IL-8, IL-17 and TNF-α in the differentconcentration PCs groups (P < 0.01) (Figure 4); nevertheless, huge significant difference was found among the differentconcentration PCs groups (P > 0.05).


Figure 2. Expression Process of CD36 on Erythroid Cells of Different Origins (A–C) Representative flow cytometry profiles showing expression of CD36 on GPA+ cells derived from hCB-CD34+ HSPCs (A), H1/AGM-S3 co-culture (B), and day-10 + 5 and 10 + 9 suspension cultures (C).

The surface markers of adult HSPC-derived erythroblasts have been well studied (Chen et al., 2009). Our flow cytometry data were consistent with previous reports that as human CD34+ HSPCs differentiate toward erythroblasts, the expression of CD34 disappeared exclusively before gaining expression of erythroid lineage markers such as GPA, CD71, CD36, and CD47 (Figures 2A and S2A). To elucidate the expression process of surface markers on hPSC-derived erythroblasts in detail, we detected erythroid-lineage-related markers established for adult-type erythroblasts, including GPA, CD71, CD47, CD81, EPO-R, CD117 (c-Kit), CD45, and CD34 (Figure S2B). Among these, the expression of CD36 on hESC-derived erythroblasts was obviously different from that on human cord blood (hCB)-CD34+ HSPC-derived erythroblasts (Figures 2A and 2B). Throughout the co-culture period, CD36 was only expressed at a low level on a small proportion of hPSC-derived GPA+ cells. In humans, CD36 is a determinant marker of committed erythroid progenitors that is expressed continuously on normal immature erythroblasts, and its expression decreases gradually with their maturation (Okumura et al., 1992). In our study, when the erythroblasts were expanded by replating day-10 co-cultured cells, the cell number of the GPA+CD36low (abbreviated as G+36low) cell fraction gradually increased and decreased again, similar to the CD36 expression process on hCB-CD34+ HSPC-derived erythroblasts (Figures 2C–2E). To further investigate the expression of CD36 during maturation of hPSC-derived erythroblasts, we recultured day-12 H1/AGM-S3 co-cultured cells in semi-solid medium, which favors optimal colony formation (with the addition of six cytokines). After 14 days, seven BFU-E colonies (1–5 × 10^5 cells per colony) were randomly chosen and subjected to flow cytometry analysis individually. In each colony, the majority of GPA+ cells were CD36+ (90.3% ± 8.2%) (Figure S2C). Day-10 co-cultured cells were replated in suspension culture. The percentage and absolute number of G+36low/+ cells increased after 3 days (day 10 + 3) and gradually decreased after an additional 4 days (day 10 + 9) (Figures 2C–2E). These data imply that early erythroid progenitors derived from hPSC/AGM-S3 co-culture seldom expressed CD36. However, the CD36 expression was upregulated when co-culture-derived HSPCs developed to a mature stage, and was gradually downregulated when they matured further, similar to hCB-CD34+ HSPCs. With the same efficiency as they were generated from the H1 cell line, erythroblasts could also be generated from other hPSC lines by co-culture with AGM-S3 cells (Figure 2F). CD36 was expressed at an extremely low level on KhES-3- and 201B7-derived GPA+ cells in co-culture, similar to its expression on H1/AGM-S3 co-culture-derived erythroblasts (Figure 2F). This result clearly demonstrates that low or negative expression of CD36 on early erythroblasts is a common phenomenon in early erythropoiesis from hPSCs.


Figure 4 Modular analysis of PPI network (A) Module 1 contains 23 genes. (B) Module 3 contains 17 genes. (C) GO and KEGG analysis of genes in Module 1. (D) GO and KEGG analysis of genes in Module 3.

Since the PPI network constructed by us was relatively small, the MCODE plugin was used to calculate the modules with an MCODE score of >5 and identify the most important modules. Finally, a total of 6 modules (for detailed results, see Supplementary Excel S1: Sheet4 and Figure S1) was identified, out of which, two (modules 1 and 3) were rich in biological processes and pathways related to tumor progression. These modules were considered to be functionally important. There were 23 genes in module 1 (12 downregulated genes and 11 downregulated genes), as shown in Figure 4A. There existed a total of 17 genes in module 3 (13 upregulated genes and 4 downregulated genes), as shown in Figure 4B. GO analysis of module 1 signified that the main enrichment was in ubiquitin–protein transferase activity. KEGG analysis showed that the main enrichment pathway was ubiquitin-mediated proteolysis (Figure 4C). The GO analysis of module 3 implied that it was mainly enriched in cell adhesion molecule binding, insulin-like growth factor I binding, and growth factor binding. KEGG analysis alluded that the main enriched pathway was the p53 signaling pathway, and PI3K-Akt signaling pathway (Figure 4D). According to module analysis, it appears that ubiquitinase may play an important role in sunitinib resistance.


Figure 4. Validation of microarray data from siWT1 and shWT1 knockdown experiments by Q-RT-PCR. All Q-RT-PCR data are displayed relative to a non-silencing si- or shRNA control that is set at 1. Data represent the mean of triplicates, and the standard deviations are indicated for each experiment. PCR reactions were done after transfection of siRNA, and the time points are indicated for each Q-RT-PCR. (A) WT1Wilms3 expression after treatment with siWT1-RNA. (B) Validation of GDF5, CASP7, MESCD2 and PODXL expression after siRNA-mediated WT1 knockdown in Wilms3 cells. (C) WT1Wilms2 and PODXL expression after treatment with siWT1-RNA. (D) WT1Wilms3 expression 48 h after infection with shWT1 lentiviruses (left panel). Expression data are displayed relative to a non-silencing shRNA-lentivirus control that is set at 1. Validation of JARID2 and PODXL1 expression in Wilms3 cells infected with shWT1 lentiviruses (right panel). Q-RT-PCR was performed 48 h after infection with shWT1 lentiviruses.

To gain more insight into the function of mutant WT1Wilms3 and WT1Wilms2 proteins, we performed siRNA (siWT1) knockdown analyses. First, we tested different WT1-specific siRNA oligonucleotides and found that a combination of two siRNAs directed against sequences in exon 6 and the 3′ UTR of WT1 achieved a very efficient knockdown 48 h after transfection of Wilms3 cells (Fig. 4A). As control a non-silencing siRNA was used. Gene expression profiles were generated using whole-genome microarrays. To identify WT1 responsive genes, the mean of two biological replicates was determined and the corresponding genes were selected (P < 0.05). A 1.5-fold change (fc) relative to the control and a minimal expression of 200 fluorescence intensity was used as cut-off, and we detected 388 genes with increased and 381 genes with decreased expression. The efficiency of WT1Wilms3 knockdown was examined by Q-RT-PCR, and a improvement of WT1Wilms3 expression by 82% was measured (Fig. 4A). Microarray data were validated for selected genes by Q-RT-PCR (Fig. 4B). It is evident that siWT1 knockdown caused decreased expression of GDF5 and increased expression of MESCD2, CASP7, PODXL1 in Wilms3 tumour cells. We next examined gene expression profiles after siWT1 knockdown in Wilms2 cells, and the efficiency of WT1Wilms2 knockdown was quantified by Q-RT-PCR (Fig. 4C). We identified 361 genes with increased and 422 genes with decreased expression levels. Microarray data showed a 2.7-fold downregulation of PODXL1, which was validated by Q-RT-PCR (Fig. 4C). Next we compared the siWT1 gene expression data from Wilms2 and Wilms3 tumour cells and identified a common set of 280 down- and 243 up-regulated genes (Supplementary Material, Fig. S2). These results demonstrate that the mutant WT1 proteins are engaged in regulating gene expression in Wilms tumour cells. Moreover, the data show that truncated WT1Wilms2 and extended WT1Wilms3 proteins (Fig. 1) regulate similar gene expression programmes. As this result is of great importance for the field of WT1 research, we wished to confirm our findings by another method. We thus performed lentiviral-mediated shWT1 knockdown experiments using Wilms3 tumour cells. The shWT1 targets exon 7 of the WT1 gene, and the most efficient knockdown of WT1Wilms3 expression (73%) occurred 48 h after infection (Fig. 4D). Gene expression analyses were performed under these conditions, and a scrambled non-silencing shRNA was used as control. A 1.5 fc relative to the control and a minimal expression of 200 fluorescence intensity was used as cut-off. The microarrays identified 1012 genes with increased and 1076 genes with decreased expression levels. Increased expression of PODXL and JARID2 was validated by Q-RT-PCR (Fig. 4E). Taken together our WT1 knockdown experiments show for the first time that mutant WT1 proteins do have a biological function and this is linked to gene regulation in Wilms tumour cells. The top 100 genes with decreased and increased expression are listed in Supplementary Material, Tables S1 and S2, showing the fold changes of expression relative to the scrambled control.


Figure 7 Beclin-1 silencing reduces RGC-5 viability following SD. (a) Effectiveness of Beclin-1 silencing and effect on LC3. Transfection of specific Beclin-1-siRNA or scramble non-targeting (NT) sequence at final concentration of 25 nM was performed, 48 h before SD, in 96-well plate with Lipofectamine 2000 according to the manufacturer’s instructions. In Beclin-1-siRNA-transfected cells, expression of the protein was reduced by 95% and was paralleled by LC3II reduction. Histograms show the densitometric analysis of autoradiographic bands. Data were normalized for GAPDH and expressed as percentage of control values (n ¼ 3). (b) Effect of Beclin-1 knockdown on survival of serum-deprived RGC-5 cells. Each bar represents the mean±S.E.M. (n ¼ 3) of cell viability, as assessed by the MTT assay. *P<0.05 versus NT siRNA and SD (SD, 0% FBS). MW, molecular weight

Beclin-1 silencing reduces RGC-5 viability under starvation. To investigate the effects of Beclin-1 reduction on RGC survival, cells were transfected with rat Beclin-1-siRNA 48 h before SD. Gene silencing reduced Beclin-1 expression by 95% compared with starved scrambletransfected cells as shown by western blotting analysis (Figure 7a) and led to a reduction of LC3II formation under starvation (Figure 7a) similar to the pattern observed in vivo (Figure 2). Beclin-1 silencing significantly worsened the improvement of RGC-5 viability, as assessed by 3-(4,5- dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, after 24 h of starvation (36 versus 67% of serum-starved cells; P<0.05; Figure 7b).


Figure 1. Effects of immediate treatment with eplerenone (Eple) and spironolactone (Spiro) on survival and hemodynamics in mice 3 days after myocardial infarction (MI). (A) Kaplan-Meier survival curve analyzed by log-rank test (*P < 0.05). (B) Hemodynamic measurements including left ventricular filling pressure (LVEDP), end-diastolic volume (LVEDV), and representative pressure-volume loops obtained via conductance catheter in sham-operated controls (Sham) and MI mice. Data shown as mean ± SEM (n = 6-9). *P < 0.05 versus Sham; †P < 0.05 versus placebo-treated MI (Pla-MI) controls.

Kaplan-Meier analysis demonstrated significantly improved survival at 7 days post-infarction exclusively in eplerenone-treated mice (Figure 1A). Infarct sizes were comparable across all experimental groups (Table 1). At 7 days post-MI, placebo-treated mice developed: (1) elevated left ventricular filling pressure (LVEDP); (2) increased end-systolic and end-diastolic volumes; and (3) severe LV dysfunction as evidenced by impaired ejection performance, dP/dtmax, dP/dtmin, and the load-independent contractility index (LV dP/dtmax/instantaneous pressure) (Figures 1B, 2; Table 1). Notably, high-dose spironolactone (100 mg/kg) worsened early LV dysfunction compared to the 20 mg/kg dose (LVEDP: 18 ± 1 mmHg; dP/dtmax: 3413 ± 295 mmHg/s; dP/dtmin: -2258 ± 198 mmHg/s; n=5). This group also showed significantly higher mortality (Figure 1A), with necropsy confirming acute heart failure (pleural effusion and pulmonary congestion). Consequently, all subsequent analyses used the 20 mg/kg spironolactone regimen.


Fig. 1. DLA-M decreases body weight and improves insulin resistance in HFD C57BL/6J mice. NCD and HFD (60% kcal fat) C57BL/6J mice were treated daily with water or 1 g/kg/day DLA-M by intragastric gavage for 10 weeks (n = 6–15). Effects of DLA-M treatment on the body weight gain and the calculated AUC (a), serum TG and TC content (b), fasting blood glucose and serum insulin levels (c), and calculated HOMA-IR and QUICKI values (d) are shown. IGTT (e); OGTT (f); ITT (g) results. Curves of the blood glucose levels and the calculated AUC are shown. Values are expressed as the means ± s.e.m. **, P < 0.01; ***, P < 0.001 compared with NCD. #, P < 0.05; ##, P < 0.01; and ###, P < 0.001 compared with HFD. Differences were assessed by ANOVA with Tukey's multiple comparison test.

Two mouse models of obesity—10-week-old male C57BL/6J and CD-1 mice—were fed a normal chow diet (NCD) or a high-fat diet (HFD) for 10 and 5 weeks, respectively. In agreement with previous work using an obese rat model (Xu et al., 2016), DLA-M prevented body weight loss in both animal models, but did not cause a significant change in the relative food intake of CD-1 mice (Fig. 1a and Supplementary Fig. 1a–c). Because obesity is strongly associated with insulin resistance, a typical characteristic of type 2 diabetes (T2D), we measured fasting blood glucose and insulin concentrations and calculated the homeostasis model assessment of insulin resistance (HOMA-IR: insulin × glucose ∕ 22.5) and the quantitative insulin sensitivity check index (QUICKI: 1 ∕ [log(insulin) + log(glucose)]). DLA-M treatment significantly improved insulin sensitivity in the mice and reversed the two indices to nearly normal levels (Fig. 1c and d), a result further confirmed by intraperitoneal glucose tolerance test (IGTT) (Fig. 1e), oral glucose tolerance test (OGTT) (Fig. 1f), and insulin tolerance test (ITT) (Fig. 1g) analyses in C57BL/6J mice.


Calcineurin removes activating phosphorylations from Hcm1. (A) Strains expressing the indicated Hcm1 proteins were treated with CaCl2 for 10 min, and phosphorylation was assayed by Phos-tag Western blot. Note that the top band in the 12C sample is also present in the 15A sample, suggesting that it is the result of Cdk1-independent phosphorylation. (B) Cdk1-phosphorylated Hcm1 proteins were incubated with or without CN for 45 min. Average percentage phosphorylation remaining is shown ± 1 SD (n = 3). Hcm1ΔPSIEIQ is significantly different from wild-type Hcm1 (*p < 0.05, Student's t test). Also see Supplemental Figure S1B. (C) Cycloheximide-chase assays comparing the stability of Hcm1 proteins with or without CaCl2 treatment. (D) Average data from five experiments were used to calculate half-life values (indicated in parentheses) of each protein with or without CaCl2.

Under these gel conditions, the phosphorylation status of a panel of Hcm1 mutants with different numbers of Cdk1 phosphosites is clearly distinguishable (Figure 2, A and C). In addition, we found that within 10 min of direct CN activation by the addition of CaCl2 to the growth medium, Hcm1 was mildly dephosphorylated (Figure 2D). This dephosphorylation was blocked by deletion of the CN docking site in Hcm1 (ΔP; Figure 2D), by deletion of the regulatory subunit of CN, Cnb1 (Figure 2D), or upon addition of the CN inhibitor FK506 (Figure 2E). These data demonstrate that Hcm1 is dephosphorylated by CN in vivo. Of note, Hcm1 retained some phosphorylation after CN activation (Figure 2, D and E), suggesting that CN might target only a subset of Hcm1 phosphosites. To test this, we examined CN-dependent dephosphorylation of previously characterized mutants in which all Cdk1 sites were mutated except for the eight activating Cdk1 sites (Hcm1-7N, with 7 N-terminal S/T-P sites mutated to A-P; Figure 2A) or the three destabilizing Cdk1 sites (Hcm1-12C, with 12 C-terminal S/T-P sites mutated to A-P; Figure 2A; Landry et al., 2014). Of interest, Hcm1-7N was almost completely dephosphorylated after CN activation, whereas Hcm1-12C phosphorylation was largely unchanged (Figure 3A). Hcm1 interacted with the CN-regulatory subunit Cna1 in this assay, and the interaction was eliminated upon deletion of a six–amino acid sequence on Hcm1 (PSIEIQ; Figure 2, A and B) that matches the consensus for the previously identified CN docking-site sequence (Roy et al., 2007).


Figure 4. Effect of pH on the release of methylprednisolone (MP) from the BSA633-MP nanoconjugate. The nanoconjugates were incubated in (A) PBS at pH 7.4 and (B) acetate buffer at pH 4.0 for 48 h at 37°C. After incubation, samples were eluted in a column with Sephadex G-100 gel. An amount of 0.5 ml of each fraction was collected. The released MP was detected by OD240, while albumin was detected by Alexa Fluor 633. (C) The nanoconjugates were incubated at pH 4.0 for 1, 4, 8, 12, 24 and 48 h at 37°C for a time course release assay. Values are the mean ± standard error of the mean (n=3).

Release profile of BSA633‑MP. The effects of neutral pH and relatively acidic pH on the release profile of BSA633‑MP conjugate were evaluated. As shown in Fig. 4A, we clearly see that under pH 7.4, almost nothing was released from the BSA633-MP conjugate. However, under pH 4.0, most MP molecules were released, a small unreleased portion remained which was eluted together with albumin (Fig. 4B). The OD240 values involved in the second elution peak vs. the total OD240 values (with subtraction of albumin itself) were calculated to roughly estimate the release percentage. As shown in Fig. 4C, with prolonged incubation, drug release was increased and at 48 h the release almost reached a plateau. Under pH 4.0 ~28% of MP drug was released after 48 h. These results indicated that the BSA633-MP conjugate was acid-sensitive and very stable in a neutral environment. Actually, the nanoconjugates were found to be stable for at least 3 months when stored in buffer at pH 7.4, without manifesting aggregation or degradation (data not shown).


Figure 3 Percentage of patients achieving glycaemic control according to HbA1c intervals. HbA1c, glycated haemoglobin; T2DM, type 2 diabetes mellitus

Finally, the analysis of the evolution of the attained glycaemic control according to different HbA1c intervals also showed that there were many remarkable changes among years in any case (figure 3). Of note, the group of patients who were less likely to achieve the corresponding glycaemic target included those younger than 65 years, without comorbidities, and duration of T2DM≤15 years (range 50.8–55.1%) (see online supplementary table S1).


Fig. 1. Hepatic insulin receptor substrate and PI3K proteins expression in ApoE knock-in mice. (A) Western blot analysis of insulin receptor substrate-1 (IRS1), PI3K/p85 and PI3K/p110 levels in the liver of ApoE3 and ApoE4 mice at 12 and 72 weeks of age. β-actin was immunoblotted to ensure similar gel loading of the starting material in each sample. The blot is a representative of three independent experiments. Densitometry analysis of total (B) IRS1, (C) PI3K/p110 and (D) PI3K/p85 level relative to β-actin level in 12 and 72 weeks old ApoE3 (white bar) and ApoE4 (grey bar) mice was performed using the NIH ImageJ software. Each value represents the mean ± SEM for individual mouse liver sample (n = 3 at each time point for each mouse line). Lower IRS1, PI3K/p85 and PI3K/p110 levels were detected in ApoE4 mice at 72 weeks of age as compared to ApoE3 mice of similar age. (*p < 0.03; **p < 0.008, using Student’s t-test.)

At 72 weeks, insulin receptor substrate 1 (IRS1) expression does not differ between fasting ApoE3 and ApoE4 mice (Fig. 1A). At 12 weeks, however, IRS1 expression is non-detectable in the fasting ApoE4 KI mice. We then examined the expression of the catalytic subunit (p110) and the regulatory subunit (p85) of phosphatidylinositol 3-kinase (PI3K). At 12 weeks, PI3K/p110 and PI3K/p85 expression were comparable between ApoE3 and ApoE4 mice. In the aged (72 weeks) fasting ApoE4 mice, however, PI3K/p110 and PI3K/p85 expression were reduced by 70% (Fig. 1C) and 44% (Fig. 1D), respectively, as compared to fasting ApoE3 mice.


Figure 1: MRTFA KO mice have smaller body weight, reduced bone mass with shorter femurs and tibiae. WT (n = 7) and MRTFA KO (n = 7) female mice (24 weeks old) were euthanized and then weighed. The image of a matched pair of WT and MRTFA KO mice is shown in (A). The femurs and tibiae of the mice were then dissected (images shown in C) and the bone lengths were measured with a caliper. The upper and lower black lines in C mark the length of the WT mouse femurs to show the differences. Whole body weights (B), femur lengths (D), and tibia lengths (E) of the mice are shown in bar graphs. Representative 3D images of cortical and trabecular region of WT and MRTFA KO mice femurs generated by μCT analysis are shown in F. Cortical thickness of WT and MRTFA KO femurs are shown in bar graphs (G). Trabecular bone volume fraction (H), trabecular number (I), trabecular thickness (J), trabecular separation (K), and connectivity density (L) are shown in bar graphs. Data are expressed as mean ± SEM. *p < 0.05; **p < 0.01.

Six-week-old MRTFA KO mice are taller and weigh less than WT littermates (Figure 1A and B and Supplementary Figure 1). This observation suggested that there might be a defect in skeletal development. To investigate a possible role of MRTFA in bone formation, lengths of the femurs and tibiae were measured with calipers. The KO mice have significantly shorter femurs and tibiae (Figure 1C, D, E and Supplementary Figure 1) when compared to WT controls. Micro-computed tomography (μCT) was performed on femurs of 24-week-old female mice (n = 7 in each group) to assess bone growth and development. μCT of the mid-diaphyseal region of femurs showed that cortical thickness of femurs from MRTFA KO mice is significantly thicker than WT controls (Figure 1F, G); however, cortical bone volume fraction (BV/TV) did not differ significantly (Table 1). μCT of the trabecular regions of proximal femurs showed a marked reduction in trabecular bone mass in the MRTFA KO mice as compared to controls (Figure 1F). Trabecular bone volume fraction is about 50% higher in KO mice (Figure 1H). Bone volume of the trabecular region is also significantly reduced, although the total tissue volume did not appear to differ (Table 1). Furthermore, average trabecular number (Figure 1I) and trabecular bone connectivity density (Figure 1L) are both lower in MRTFA deficient mice, and trabecular spacing (Figure 1J) is larger. There was no significant difference in trabecular thickness (Figure 1K), which is likely due to high bone turnover rate in rodents.


The c-Myb protein level is critical for GSK3β-dependent cell survival. (A) Jurkat, K562, and RPMI 8226 cells were treated with or without 5 or 10 μM SB216763 for 8 h, and cell lysates were analyzed by immunoblotting. (B) RPMI 8226, K562, and Jurkat cells were infected with lentivirus expressing GSK3β shRNA or a nonspecific control shRNA. Four days after infection, cells were harvested for immunoblotting analysis. (C) Jurkat cells were infected with lentiviral-overexpressed c-Myb or a control vector and after 48 h treated with or without SB216763 (10 μM) for another 2 or 3 days. Cell numbers were determined at days 0, 2, and 3 with a CASY Model TT cell counter (left). Data are represented as mean and SD. The overexpression of c-Myb was verified by immunoblotting (right). (D) Jurkat cells were treated with cycloheximide (20 μg/ml) in the absence or presence of LiCl (10 mM) for the indicated time points, and cell lysates were analyzed by immunoblotting. For the quantification on the right, the c-Myb protein level at time 0 was set at 100%. (E) Jurkat cells were treated with or without SB216763 (10 μM) or LiCl (10 mM) for 6 h, after which MG132 (10 μM) was added for another 4 h. c-Myb and nonspecific control immunoprecipitations were analyzed by SDS–PAGE and immunoblotting.

We next tested the effect of GSK3β inactivation on the c-Myb and LEF-1 proteins. Of importance, in all leukemia cells examined, the levels of c-Myb were increased upon SB216763 or LiCl treatment, whereas the levels of LEF-1 did not show significant similarity (Figure 4A and Supplemental Figure S3). RNAi-mediated depletion of GSK3β in all three kinds of leukemia cell lines led to an obvious decrease in c-Myb and upregulation of cleaved caspase 3 (Figure 4B). c-Myb overexpression could rescue the inhibition of cell proliferation by SB216763 in Jurkat cells, demonstrating that c-Myb is a critical growth-regulatory target of GSK3β kinase activity in these cells (Figure 4C). Inhibition of de novo protein synthesis by cycloheximide (CHX) showed that c-Myb degraded faster in cells treated with SB216763 or LiCl (Figure 4D and unpublished data). In addition, we found that the ubiquitination of c-Myb was increased after GSK3β inactivation (Figure 4E).