Figure 4. Analysis of three donors indicate that data obtained from PSMS vs. PBMC generated compensation matrices are similar. A:Mean and standard deviation of negative and positive populations for each T cell panel marker using compensation matrices generated from PSMS and PBMC. B: X-Median (Median fluorescence intensity) of three donors PBMC stained with panel specific markers given the compensation matrix generated by PSMS and PBMC. C: SI of PBMC from three donors using PSMS and PBMC matrices.

To determine the effects of compensating with PSMS versus PBMC we studied three donors in which we compared the data on a global scale looking at the percent negative and positive populations (Fig. 4A), the MedFI of both of these populations (Fig. 4B) and SI (Fig. 4C). For all three parameters (autofluorescence, population distribution, and SI) no major significant differences were detected when using either PSMS or PBMC compensation matrices (Figs. 4A–4C). The only significant difference detected was the X MedFI of the negative fraction of FL9 (Pacific Blue 450/50 BP), on average the X MedFI of data using the PBMC based compensation was 3.07 lower than data compensated using PSMS. While this difference was observed, it is important to note that this difference did not affect the population distribution of the sample set (Fig.4A). There were large differences in the standard deviation of FL6 (APC 660/20 BP) and FL8 (Pacific Blue 450/50 BP) MedFI of the positive cell population. However, this result was independent of the compensation used and reflects donor variability. Perhaps the most striking difference between PSMS and PBMC compensation matrices were between FL7 (APC-A700 725/20 BP) and FL8 (APC-A750 775 LP). This is important to note as FL7 corresponds to IL23R APC-A700 staining. This staining using PBMC did not yield a distinct positive and negative fraction thus making compensation difficult.


Figure 5 Suppression of DNA damage induced by pressure overload in HMGB-1 Tg mice. (A) Left-ventricular transverse sections in WT and HMGB1-Tg mice at 4 weeks after TAC, and heart weight: body ratios in TAC or sham-operated mice. Data are mean±SEM from eight mice for each group. *P < 0.05 and **P < 0.01 vs. sham-operated mice of the same strain; †P < 0.05 vs. TAC-operated Tg mice. (B) Quantitative analyses of ANP, BNP, and b-MHC gene expression in WT and HMGB1-Tg mice at 4 weeks after TAC. Data are mean±SEM from eight mice for each group. *P < 0.05 and **P < 0.01 vs. sham-operated mice of the same strain; †P < 0.05 vs. TAC-operated Tg mice. (C) Data showing echocardiographic measurements in WT and HMGB1-Tg mice at 4 weeks after TAC or sham surgery. IVS, interventricular wall thickness; LVEDD, left-ventricular end-diastolic dimension; LVESD, leftventricular end-systolic dimension; %LVFS, left-ventricular fractional shortening. Data are mean±SEM from eight mice for each group. *P < 0.05 vs. sham-operated WT mice; †P < 0.05 vs. TAC-operated WT mice. (D) Survival curves in WT and HMGB1-Tg mice after TAC.

At 4 weeks after the TAC operation, the increase in the weight of the heart was significantly higher in HMGB1-Tg mice than in WT-mice (Figure 5A). We next examined the mRNA expression of foetal cardiac genes after TAC. The expressions of ANP, BNP, and b-MHC were significantly up-regulated in the TAC group compared with the shamsurgery group, and this upregulation was significantly intensify in HMGB1-Tg mice (Figure 5B). Moreover, systolic dysfunction and leftventricular dilatation after TAC were attenuated in HMGB1-Tg mice compared with WT-mice (Figure 5C, Supplementary material online, Table). Furthermore, the survival rate after TAC was significantly lower in HMGB1-Tg mice than in WT mice (Figure 5D).


To examine the effect of rapamycin on the expression of miR-30a and autophagy of VSMcs, the expression of miR-30a and Beclin1 was determined by RT-qPcR. The results demonstrated that in aging cells miR-30a was significantly downregulated while Beclin1 was significantly downregulated compared with young cells (P<0.01; Fig. 2A and B), indicating that both miR-30a and Beclin1 might be associated with senescence in VSMcs. However, when treated with rapamycin, miR‑30a was significantly downregulated in both young and aging cells (P<0.05; Fig. 2A). Additionally, the expression of Beclin1 was significantly upregulated in both young and aging cells following rapamycin treatment (P<0.05; Fig. 2B). These results clearly demonstrated that rapamycin inhibited miR-30a expression while it induced Beclin1 expression.

Rapamycin inhibits senescence in VSMCs and alleviates cell cycle arrest. SA-β-gal staining was conducted to examine the effect of rapamycin on senescence of VSMCs. As illustrated in Fig. 1A and B, the ratio of SA-β-gal-positive cells was significantly higher in aging cells compared with young cells (P<0.01). However, following rapamycin treatment, the ratio of SA-β‑gal‑positive cells significantly decreased in aging cells (Fig. 1B), indicating that rapamycin could inhibit senescence in VSMCs. Flow cytometry analysis revealed that G1 arrest occurred in aging cells and this cell cycle arrest was reduced when cells were treated with rapamycin (Fig. 1C and D). Further analysis of p16, p21, p53 and SA-β-gal expression levels by western blotting demonstrated that rapamycin treatment promoted the increasing levels of senescence-related proteins in aging cells (Fig. 1E and F). These results suggested that rapamycin could inhibit senescence of VSMCs through inhibition of cell cycle arrest and senescence-related proteins.


Effect of curcumin and resveratrol on ASK1-overexpressed cells viability. LNCaP and PC-3 cells were transiently transfected with vector pcDNA3.1 (MOCK), wild-type ASK1(ASK1) or kinase inactive mutant (KM-ASK1). Cells were set overnight and then treated with 25 µM curcumin (A) or 50 µM resveratrol (B). Cell viability was studied using MTT assay after 24 treatment. Data are shown as mean ± S.E.M. of 6 independent samples. *p < 0.05; **p < 0.01; ***p < 0.001 versus CON. PC-3 cells (C) transiently transfected with vector, ASK1 or KM-ASK1 were incubated with or without curcumin (25 µM) for 24 h. Apoptosis was evaluated after staining with Annexin V/PI by flow cytometry.

The effects of four bioactive compounds—melatonin, silibinin, curcumin, and resveratrol—on the viability and proliferation of prostate cancer cells were evaluated using the MTT assay (Fig. 1A). After 48 hours of treatment, growth of androgen-sensitive LNCaP cells was significantly inhibited by melatonin at 200 µM and 1 mM and by silibinin at 50 µM, while no significant effect was observed in androgen-insensitive PC-3 cells (Fig. 1A). Curcumin and resveratrol inhibited the viability of both cell lines at concentrations above 10 µM and 60 µM, respectively. Since the MTT assay cannot distinguish between growth inhibition and cell death, annexin V staining combined with flow cytometry was performed after culturing LNCaP and PC-3 cells with melatonin (1 mM), silibinin (50 µM), curcumin (25 µM), or resveratrol (50 µM) (Fig. 1B). Only curcumin and resveratrol treatments induced a significant increase in apoptotic cells in both cell types. To confirm this, western blot analysis assessed levels of the anti-apoptotic protein BCL2 and the pro-apoptotic protein BAX. In LNCaP cells, curcumin and resveratrol treatment caused a clear decrease in BCL2 and an increase in BAX protein levels. Conversely, melatonin incubation increased BCL2 levels, and silibinin treatment showed no significant changes in the BCL2/BAX ratio (Fig. 1C). Figure 1D shows morphological changes in LNCaP and PC-3 cells after 48 hours of incubation with the compounds. Typical apoptotic features—such as cell shrinkage, rounding, and partial detachment—were observed following curcumin treatment in both cell types. PC-3 cells treated with resveratrol also showed apoptotic morphology preceded by cytoplasmic vacuolization. Melatonin and silibinin treatments induced significant morphological changes including long processes (neurites) and rounded nuclei, characteristic of neuroendocrine differentiation, consistent with previous reports.


Fig. 4. Mechanism dissection studies on interleukin-6 (IL-6) regulation of hypoxia-inducible factor (HIF) following cisplatin treatment. (a) Hypoxia response element–luciferase (HRE-luc) assay. HEK293 cells were transfected with HRE-luc-containing plasmids and incubated with various amounts of IL-6. After 24 h of incubation, luciferase activities were measured. (b) HRE-luc assay. A549IL-6si ⁄sc and H157IL-6si ⁄sc pairs were transfected with equal amounts of HRE-luc-containing plasmids. After 24 h of transfection, luciferase activities were measured. (c) Prolyl hydroxylase (PHD) mRNA levels in A549IL-6si ⁄sc and H157IL-6si ⁄sc cell sets, with or without cisplatin treatment. Cells were either non-treated or treated with cisplatin (5 lM, 72 h), total RNAs extracted, cDNA converted, and mRNA expressions were analyzed. (d) Western blot analysis of von Hippel-Lindau disease tumor suppressor (VHL), HIF1a, and HIF2a levels in A549IL-6si ⁄sc and H157IL-6si ⁄sc cell sets, with or without cisplatin treatment, in the presence or absence of MG132. Cells were either non-treated or treated with cisplatin (5 lM, 72 h) in the absence or presence of MG132 (10 lM), cell extracts were obtained, and HIF1a and HIF2a levels were analyzed in Western blot. (e) Summary of the results of mechanism studies. *P < 0.05; **P < 0.01; ***P < 0.001. CSC, cancer stem cell.

We observed an decrease in HRE-luc activities with IL-6 addition in both cell lines (Fig. 4a), suggesting the IL-6 regulation of HIFs at the transcriptional level. We then investigated HRE-luc activities in A549IL-6si ⁄sc and H157IL-6si ⁄sc cell sets after cisplatin treatment. Interleukin-6-expressing sc cells showed higher HRE-luc activities than IL-6si cells (Fig. 4b), implying IL-6-mediated upregulation of HIFs at the transcriptional level may contribute to cisplatin resistance. We next tested whether the IL-6-mediated upregulation of HIFs on cisplatin treatment is also due to inhibition of HIF degradation. We tested whether the levels of PHDs that are known to be associated with VHL-mediated HIF ubiquitination are different in IL-6si ⁄sc cells after cisplatin treatment. When we investigated mRNA levels of PHDs in A549IL-6si ⁄sc and H157IL-6si⁄sc cell sets, we found the PHD levels were significantly higher in IL-6si cells than in sc cells, and that the difference was more significant in the presence of cisplatin (Fig. 4c). The upregulation of HIFs after cisplatin treatment observed in sc cells was abolished when cells were treated with the proteasomal degradation inhibitor MG132 (Fig. 4d). However, we found that the VHL level was not affected by IL-6. These results suggest that IL-6 triggers the promotion of ubiquitination by mediating PHD level decrease after cisplatin treatment. Figure 4e summarizes these results.


Figure 1. The combination of AT406 and upregulation of c-FLIPL/S by c-FLIP-siRNA sensitizes the osteosarcoma cells U2OS to TRAIL-induced apoptosis. (A) The osteosarcoma cells U2OS were treated with different concentrations of TRAIL for 24 h. In three other concentration-response experiments, cells were pre-treated with AT406 (A) for 1 h and with or without transient transfection with c-FLIP-siRNA for 24 h. The cell viabilities were determined by an MTT assay. The results are shown as the mean ± SEM of three separate experiments. (B) Caspase activity of U2OS cells in response to TRAIL with or without pre-treatments of A and c-FLIP-siRNA as indicated in the graph was measured by a Caspase-Glo 3/7 assay. (C) The U2OS cells were treated with TRAIL for 5 h, and with or without pre-treatment of A for 1 h, and transient transfection with c-FLIP-siRNA for 24 h as indicated in the graph. The extrinsic apoptosis related proteins were determined by western blots and statistical analyses and are shown in (D). In all experiments if not specified: A, AT406 at 1 µg/ml; EV, control empty vector; c-FLIP-siRNA, pSuper-puro-siRNA-c-FLIP vector; T, TRAIL at 100 ng/ml. The results are shown as the means ± SEM in all bar graphs of western blots of three independent experiments (NS>0.05, ***p<0.001).

Inhibition of c-FLIPS/L and IAPs can increase the sensitivity of U2OS to TRAIL-induced apoptosis. By evaluating TRAIL promotion of tumor cell apoptosis, we found that U2OS is resistant to TRAIL-induced apoptosis. Because c-FLIP is a crucial apoptotic resistance factor, we used the c-FLIP-siRNA overexpression plasmid which was reported in our previous study to inhibit the expression of c-FLIP (Fig. 1A) (27). The results of an MTT assay showed that in combination with TRAIL with or without c-FLIP-siRNA, AT406 could increase the cell death rate of U2OS (Fig. 1A). Results of the Caspase-Glo 3/7 assay (Fig. 1B) confirmed that c-FLIP-siRNA in the presence of AT406 activated caspase proteins in the TRAIL-activated apoptotic pathway. That is, apoptosis initiator protein caspase-8, apoptosis executor protein caspase-3 and apoptosis substrate poly(ADP-ribose) polymerase (PARP) were all in their cleaved states to various degrees (Fig. 1C and D). All of these results demonstrated that c-FLIP-siRNA and AT406 together activated the extrinsic apoptotic pathway in the presence of TRAIL to induce apoptosis.


FIGURE 4 Statistical graph of systolic blood pressure (a), diastolic blood pressure (b), heart rate (c), and T3 phase tilt angle (d) before and after the application of nitroglycerin. All data arepresented as mean ± SD. *p < .05, ****p < .0001

The application of nitroglycerin did show a significantly antihypertensive function in both systolic blood pressure (163.8 ± 17.1 mmHg vs. 182.0 ± 12.1 mmHg, p < .0001) and diastolic blood pressure (92.7 ± 11.8 mmHg vs. 74.0 ± 9.6 mmHg, p < .0001) as shown in Figure 4A and 4B5. The heart rates of patients increased significantly after the application of nitroglycerin (77.3 ± 11.0 vs. 92.6 ± 10.3, p = .023) (Figure 4C). The change of the T3 phase tilt angle before and after the application of the antihypertensive medication was obvious. After the application of nitroglycerin, the inclination angle of T3 increased significantly (-50.6 ± 16.2° vs. −25.6 ± 21.9°, p < .0001) (Figure 4D).


**Figure 4. Bid Phosphorylation on Serine 66 Sensitizes Cells to Apoptosis during Mitotic Arrest**(A) RKO cells stably infected with pVenus, pVenus-shBid, or pVenus-shBid coexpressing the indicated mouse (left panel) and human (right panel) BidYFP variants under the ubiquitin promoter were analyzed by immunoblotting with an antibody that recognizes both human and mouse Bid. Immunoblotting for Erk was used as a loading control. Endogenous human Bid is only present in the control cells.(B) The control RKO lines and those expressing mouse BidYFP variants were untreated or treated with 1 μM paclitaxel for 18 hr. Lysates were analyzed by immunoblotting for Bid and active caspase 3. Erk was a loading control. Note the shift in mobility of BidYFP-WT and BidYFP-G94E in paclitaxel-treated RKO cells.(C) The RKO lines from (A), untreated or treated with 1 μM paclitaxel for 18 hr, were immunostained for active caspase 3 and apoptosis quantified. The data represent the mean of three independent experiments. The error bars represent SEM. Data were analyzed by ANOVA.(D) Images of the paclitaxel-treated RKO cell lines from (C), immunostained for active caspase 3. Nuclei were stained with Hoechst.(E) Bid⁻/⁻ MEFs, infected with the indicated pVenus lentiviruses, were left untreated or treated with 1 μM paclitaxel. Apoptosis was quantified as above. The data represent the mean of three independent experiments. The error bars represent SEM. Data were analyzed by ANOVA.(F) RKO cells infected with the indicated lentiviruses expressing human Bid or human BidS67A were treated with paclitaxel as in (C). Cells showed similar responses to those expressing the mouse BidYFP. The data represent the mean of three independent experiments. The error bars represent SEM. Data were analyzed by ANOVA.(G) The indicated RKO lines, untreated or treated with monastrol for 18 hr, were immunostained for active caspase 3 and apoptosis quantified. The data represent the mean of three independent experiments. The error bars represent SEM. Data were analyzed by ANOVA.

To test if Bid-pS66 regulates apoptosis during mitotic arrest, we generated stable RKO lines where endogenous hBid was knocked down and substituted by mouse BidYFP-WT, BidYFP-S66A, BidYFP-S66D, or BidYFP-G94E. As expression of mBidYFP was signiﬁcantly higher than endogenous hBid using the original pVenus vector with an EF1a promoter (Figure S3A), we replaced it with an ubiquitin (Ub) promoter. This led to expression of mBidYFP at levels comparable to endogenous hBid (Figures 4A and S3B). When the RKO lines were treated with paclitaxel for 18 hr, although BidYFP-WT rescued apoptosis following endogenous Bid knockdown, neither Bi-dYFP-S66A nor BidYFP-G94E BH3 mutant were able to restore the response (Figures 4B–4D). Notably, BidYFP-S66D was not a functional phospho-mimetic and was also unable to restore the response. Different results were obtained in Bid-/-MEFs stably expressing Ub-promoter-driven BidYFP-WT, BidYFP-66A, and BidYFP-G94E (Figure 4E). To ask if phosphorylation of human Bid on S67 had the same role, we generated RKO cells where endogenous hBid was knocked down and hBidYFP-WT or hBidYFP-S67A expressed (Figure 4A). hBidYFP-WT rescued paclitaxel-induced apoptosis in Bid knockdown RKO cells, but hBidYFP-S67A did not (Figures 4F and S4A). To determine whether the proapoptotic role of Bid during mitosis was seen when cells were treated with other antimitotic drugs, we treated RKO cells with monastrol. These cells also displayed Bid-S66-phosphorylation-dependent apoptosis (Figure 4G), although the level of cell death was much lower than with paclitaxel.


Figure 2. The MENIN/MLL2 H3K4 Methyl Transferase Is Cell-Cycle Regulated and Controls the Activation of Bivalent Genes in G1(A) qChIP of Fucci hESC cell-cycle fractions examining levels of MLL2 and WDR5 on the GATA6 and SOX17 promoters. Data are the average of three independent replicates.(B) qChIP assays for MLL2 in untreated or MI-2 (25 μM for 24 hr)-treated WA09 ESCs at the indicated promoters. Data are the average of three independent replicates.(C) qChIP assays for MLL2 in ESCs of pluripotency genes. Data are the average of three independent replicates.

. To determine whether CDK2 activity was required for MLL2 recruitment to bivalent genes, we added the inhibitor CVT-313 (Brooks et al., 1997) for a brief period (4 hr) so as not to impose a cell-cycle block, and no major perturbations in cell-cycle progression were observed during this time (Figure 3A). The addition of CVT-313 suppressed developmental transcripts had no effect on OCT4 transcripts or transcripts for non-cell-cycle-regulated bivalent genes, and significantly increased MLL2 recruitment to developmental genes (Figures 3B, 3C, and S3C). MENIN binding, however, was not affected by CDK2 inhibitor treatment (Figure 3C).


Figure 1 experimental design and outcome.Notes: (A) Mouse lewis lung carcinoma llc1 cells (1×106) mixed with Matrigel were injected subcutaneously in c57Bl/6 mice on day 0. The mice were randomly assigned into five treatment groups (a–e). Treatment started on day 1, with the doses and schedules indicated by arrows along the time course. (B, C) The results (tumor weight) of the first and second experiments, respectively. Data represent means ± seM (error bars). The number of animals per group is indicated under each group.Abbreviations: iP, intraperitoneally; PO, per os (by mouth); iU, international unit; seM, standard error of the mean.

ResultsiFnγ and celecoxib inhibit mouse lung-tumor growthWe implanted mouse LLC1 cells subcutaneously into syngeneic C57BL/6 mice. The mice were randomly assigned into five groups: a) treated with saline as the placebo control group, b) treated with IFNγ, c) treated with celecoxib (COX-2 inhibitor), d) treated with a combination of IFNγ and celecoxib, and e) treated with zoledronic acid that can kill macrophages (Figure 1A). In the first experiment, we found that IFNγ alone reduced tumor weight by 30% compared to the control group (P < 0.01, Figure 1B). Celecoxib alone reduced tumor weight by 27% compared to the control group (P<0.01, Figure 1B). The combination of IFNγ and celecoxib reduced tumor weight by 23% compared to the control group (P < 0.01, Figure 1B). However, there was no statistically significant difference between the combinedtreatment group and IFNγ-alone or the celecoxib-alone groups (P<0.05, Figure 1B). Zoledronic acid did not significantly reduce tumor weight compared to the control group (P<0.05, Figure 1B). In the second experiment, we found that IFNγ alone reduced tumor weight by 50% compared to the control group (P<0.01, Figure 1C). Celecoxib alone reduced tumor weight by 31% compared to the control group (P<0.01, Figure 1C). The combination of IFNγ and celecoxib reduced tumor weight by 34% compared to the control group (P<0.01, Figure 1C). Like the first experiment, there was no statistically significant difference between the combinedtreatment group and IFNγ-alone or celecoxib alone groups (P<0.05, Figure 1C). Unlike the first experiment, zoledronic acid reduced tumor weight by 16% compared to the control group (P<0.01, Figure 1C). Of note, we found that all animals gained body weight during the treatment, and there was no statistically significant difference among the five groups (data not shown).


FRAP analysis of PRC1 fusion proteins binding to interphasic and mitotic chromatins. (A) Representative FRAP images of YFP-Cbx2 fusion protein at metaphasic and interphasic chromatins of ES cells. The images were taken before (pre) and after (post) photobleaching. The bleaching area is indicated and outlined in white. (B–I) FRAP curves of PRC1 fusion proteins at interphases and metaphases of PGK12.1 ES cells. The FRAP curves are the normalized  fluorescence intensities of the bleached areas as a function of time after photobleaching and are an average of at least eight cells. Error bars indicate SDs of means. (J) Immobile fraction of PRC1 fusion proteins at interphasic and metaphasic chromatins. The immobile fraction was calculated from FRAP curves by fitting a first-order kinetic model. The dashed line indicates the contrast between interphase and metaphase. Data are an average of at least eight cells. (K) Residence time of PRC1 fusion proteins at interphasic and metaphasic chromatins. The residence time was calculated from FRAP curves by fitting a first-order kinetic model. The bar with NA indicates that the fusion protein is immobilized at mitotic chromatin, and thus the residence time is not measurable within the time scale of the experiments. The dashed line indicates the contrast between interphase and metaphase.

Figure 5, A–I: The mCherry-H2A fusion protein served as a guide for placing bleach spots at mitotic chromosomes. Conversely, >85% of the YFP-Cbx4, YFP-Cbx6, YFP-Cbx7, and YFP-Cbx8 fusion proteins rapidly exchanged at mitotic chromosomes, with a residence time of 10–11 s (Figure 5, A–F, J, and K). During interphase, >90% of the Cbx-family fusion proteins showed fluorescence recovery, with a residence time of ∼10–13 s (Figure 5, A–F, J, and K), consistent with previous studies. Analysis of FRAP curves of mitotic chromosomal binding of Cerulean-Ring1b, Cerulean-Phc1, and Cerulean-Mel18 fusion proteins revealed that nearly 50% of these fusion proteins stably bind to mitotic chromosomes without exchange (Figure 5, G–K), which is similar to YFP-Cbx2 but differs from other Cbx-family fusion proteins. The FRAP curves were normalized and plotted as described in Figure 5. The mCherry-H2A was used to mark mitotic chromosomes. Scale bar, 5 μm. (C) FRAP curves of interphases and metaphases of YFP-Cbx2 and its variant fusion proteins expressed in HeLa cells. FRAP analysis is described in Figure 5. (D) Residence time and immobile fraction of YFP-Cbx2 variant fusion proteins at interphasic and mitotic chromatins.


Decitabine-mediated induction of CT45 expression in EOC cells. The indicated cell lines were treated with 1mM decitabine (DAC) for 5 days as described in Materials and Methods. (A) CT45 mRNA expression and CT45 promoter methylation after vehicle (PBS) or DAC treatment. CT45 mRNA expression was determined by RT-qPCR and CT45 promoter methylation was determined by pyrosequencing (average of Region 1 C Region 2 methylation). Data represent mean C/– SD. The two-tailed paired t-test p-value is indicated (*:P < 0.05; **: P < 0.01; ****: P < 0.0001; NS: not signiﬁcant). (B) Western blot analysis of CT45 protein expression after vehicle (PBS, –) or DAC (C) treatment. b-actin and FastGreen staining are shown as loading controls.

The primary tumor data presented above establishes a correlation between CT45 promoter hyperhmethylation and CT45 expression, but does not demonstrate causation. To establish a mechanistic connection, we treated EOC cell lines with varying baseline CT45 expression levels using the DNA methyltransferase (DNMT) inhibitor decitabine (DAC), a cytosine analog that specifically inhibits DNA methylation by covalently trapping DNMTs to DNA. Following DAC treatment, the CT45 promoter was hypomethylated in all treated cell lines (Figure 9A). Notably, CT45 expression was robustly promoted in low-expressing cell lines (IOSE121, OVCAR3, SNU119), with less or no induction in two lines with higher basal CT45 expression (A2780, OVSAHO) (Figure 9A). Importantly, CT45 protein was also promoted at varying levels in EOC cell lines treated with DAC (Figure 9B).


FIGURE 2 Diet effects on peripheral metabolism. A-C) Total cholesterol, low-density lipoprotein (LDL), and high-density lipoprotein (HDL) levels were significantly reduced following the Mediterranean diet (Med-diet) and increased following the Western diet (West-diet) for both normal cognition (NC) and mild cognitive impairment (MCI) groups (all Ps < .001). D, Plasma glucose levels increased for the NC group and decreased for the MCI group following the West-diet (P = .01), with no changes observed for groups following the Med-diet. E Plasma insulin levels increased for the NC group and decreased for the MCI group following Med-diet (P = .031). Insulin also tended to decrease for the MCI but not the NC group during the West-diet (P = .051). F, The NC group showed increased HbA1c levels following the West-diet, and the MCI group showed decreased levels (P = .049). Figures depict change scores for ease of interpretation, adjusted means from repeated measures analysis of covariance are included in Table S2. Error bars represent ± 1 standard error from the mean. Significance is set at +P < .10, *P < .05, ** P < .01, or *** P < .001

The West-diet decreased and the Med-diet reduced total cholesterol levels for both NC and MCI groups similarly (diet x time F[1,69] = 21.89, P = .0001; Figure 2A). This pattern was also observed for LDL and HDL cholesterol (diet x time F[1,68] = 26.54, P = .0001 for LDL; diet x time F[1,72] = 13.07, P = .0006 for HDL; Figure 2B-C). Both groups' levels were unchanged following the Med-diet, whereas following the West-diet, the NC group's levels reduced and the MCI group's levels decreased (diagnosis x time F[1,34] = 7.47, P = .010; Figure 2D). For fasting insulin levels, the NC group increased and the MCI group decreased following the Med-diet; the MCI group insulin levels also decreased during the West-diet, whereas the NC group showed little change. A three-way interaction of diet x cognitive group x time was observed for HbA1c (F[1,69] = 4.67, P = .034); the NC group showed increased HbA1c levels following the West-diet, and the MCI group showed increase levels (F[1,36] = 4.17, P = .049; Figure 2F), whereas the opposite, though non-significant, pattern was observed for the Med-diet.


Figure 5: Munc18c depletion diminishes high-KD-evoked exocytosis of previously-docked insulin SGs. (A,B) Insulin SG exocytosis dynamics evoked by 50 mmol/l KCl from control (A) and Munc18c-KD (B) human beta-cells. Black and white bars indicate pre-docked and newcomer SGs, respectively. Data from 10 cells for each group, expressed as means ± SEMs. (C) Normalized cumulative fusion events of SGs per unit area from control (black circles) and Munc18c-KD (white circles) beta-cells. (D) Summary of fusion events from pre-docked and newcomer SGs in first- and second-phase after 50 mmol/l glucose stimulation. Values represent means ± SEMs. *p < 0.05.

Munc18c depletion affecting both phases of GSIS in our study seems inconsistent with a previous study showing heterozygous Munc18c-KO mice islets affecting predominantly the second-phase GSIS. Since a major portion of first-phase GSIS is attributed to predocked SGs, which are preferentially released by high-Kþ stimulation in the first few minutes, we employed this protocol on Munc18c-KD human beta-cells. Consistently, 50 mmol/l KCl triggered exocytosis of mainly previously-docked (pre-docked) SGs (black bars) in the first few minutes from control and Munc18c-KD cells (Figure 5A,B). Here, there was a 59% reduction in exocytosis in the first 5 min (Figure 5C) accounted for mostly from a reduction of exocytosis of pre-docked SGs in the Munc18c-KD cells, compared with control cells (summary analysis in Figure 5D;


C. elegans meiosis has both anaphase A and anaphase B. (A) Table of spindle lengths and distances between the centers of chromosomes measured at the start of anaphase A, the end of anaphase A, and the start of cortical ingression for both MI and MII meiotic spindles. Averages and SEM are shown. (B) Graphs depicting the change in distance between separating chromosomes in time-lapse sequences of four embryos show different rates during anaphase A and anaphase B. (C) Rates of chromosome separation from multiple time-lapse sequences of MI anaphase A, MII anaphase A, MI anaphase B, and MII anaphase B. Although there is no significant difference between MI and MII rates, the difference between all anaphase A rates and all anaphase B rates is extremely significant (****p < 0.0001). (D) Table of the averages of the rates shown in C. All rates were determined from time-lapse images of embryos expressing GFP::tubulin and mCherry::histone and are expressed as micrometers per minute.

A plot from a single embryo is shown in Figure 1C, and data from multiple embryos are shown in Figure 2. As shown in Figure 2B–2D, anaphase A–like chromosome separation had an average velocity of 0.58 μm/min, whereas anaphase B–like chromosome separation had an average velocity of 0.85 μm/min. The anaphase A–like velocities were significantly different from the anaphase B–like velocities, whereas the velocities during anaphase I were not significantly different from those during anaphase II (Figure 2C). The short length of the spindle at anaphase onset (5.84 μm; Figure 2A) suggested that chromosomes might already be in physical contact with spindle poles before anaphase onset. This distance is similar to the average spindle length at the onset of anaphase A determined from GFP::tubulin, 4.08 μm (Figure 2A).


Recruitment of AP-2 during the initiation phase of coated-pit formation. (A) Representative example of a fluorescence intensity tracing of AP-2 (σ2-eGFP) from a time series obtained from SUM-AP-2.1 imaged by TIRF microscopy. The tracing corresponds to the formation of a coated pit containing AP-2 tagged by σ2-eGFP; the time series was acquired every 150 ms with an exposure of 60 ms/frame. The inset shows the fitted steps and highlights the first and second steps. (B) Histogram of the fluorescence intensities associated with the first and second steps (gray). The best fit (dark blue) corresponds to the added contributions (orange) of one, two, and so on σ2-eGFP molecules. The data from three cells correspond to 253 first and 224 second steps detected during the initiation of 253 independent coated pits. The combined dwell time for both steps was 2.6 ± 1.9 s (mean ± SD). (C) Selection of the best fluorescently tagged AP-2 (this study) or a mixture of native AP-2 and fluorescently tagged AP-2 (Cocucci et al., 2012).

In 253 traces from three cells, the first detectable events during coated-pit initiation were two consecutive, stepwise increases in AP-2 fluorescence intensity (Figure 2A) with average dwell times of 2.9 and 8.3 s, respectively. The distribution of fluorescence intensity increments for both steps is shown in Figure 2B. Assuming full replacement of σ2 by σ2-eGFP (equivalent to a substitution fixed at 1), a search for the recruitment model with the lowest Bayesian information criterion (BIC) score (Burnham and Anderson, 2002; i.e., the model that best fit the experimental observations) yielded two AP-2 complexes per step in approximately 60% and 70% of the first and second steps, respectively (insets in Figure 2B and 2C, top, and model i). We validated the recruitment model with an independent search that included exploration for partial replacement of σ2 by the σ2-eGFP or incomplete maturation of a fluorescent eGFP (Figure 2C, bottom, and associated models).


Figure 4: Changing FXRα2/α1 ratios promotes transcriptional reprogramming. (A) Target gene expression analysis by qRT-PCR in hepatocytes expressing GFP or FXRα2 and treated with vehicle (DMSO) or 250 μM cholic acid (CA) for 24 h. Bars depict mean values and error bars represent SEM (n = 5). *p < 0.05 compared to vehicle-treated or between the indicated groups. (B) Gene expression analysis by qRT-PCR in hepatocytes co-transduced with FXRα1 and FXRα2 in different proportions (+++, 100%; ++, 70%; +, 30%; –, 0%). Graphs represent expression levels relative to hepatocytes transduced with FXRα1 alone. Graphs depict mean values and error bars represent SEM (n = 5). *p < 0.05 compared to FXRα1 alone or between the indicated groups.

Bile acids function as endogenous FXR ligands and greatly increase their capacity to transcriptionally activate a vast array of target genes. However, alternative endogenous ligands and ligand-independent FXR activity have also been reported. To investigate whether bile acids affect the gene program under FXRα2 control, we treated hepatocytes expressing FXRα2 or GFP alone with 250 μM cholic acid (CA), a concentration shown to yield moderate FXR activation. CA treatment in GFP-transduced cells increased the expression of most FXRα2 target genes involved in lipolysis, fatty acid (FA) oxidation, mitochondrial uncoupling, and ketogenesis (Figure 4A and Figure S4A). The exceptions were Atgl, Hsl, Cgi-58, and Fabp1, whose expression was not affected by CA treatment. In contrast, when FXRα2 expression was increased, only Ucp2, Hmgcs2 and to a lesser extent Ppara displayed reduced mRNA levels upon CA treatment, when compared to vehicle-treated cells. For all other genes analyzed, expression was changed or in some cases (e.g., Atgl and Slc25a47) enhanced by CA treatment. Interestingly, increased FXRα2 levels blunted the CA-mediated effects on Bhlhe41 expression. In addition to ligand-mediated activation, the relative expression of FXR splice isoforms has important implications on hepatic metabolism. To verify whether changing FXRα2/α1 ratios is sufficient to promote transcriptional reprogramming, we co-expressed different proportions of FXRα1:FXRα2 in hepatocytes. For clarity, the relative amounts of FXRα1 and α2 adenoviral particles used are illustrated in Figure 4B. While maintaining similar total FXR expression levels, increasing FXRα2/α1 ratios gradually induced the FXRα2-mediated gene program conducive to increased lipolysis, FA oxidation and ketogenesis (Figure 4B and S4B). Bhlhe41 expression showed the opposite pattern. These data indicate that changes in FXR isoform composition through alternative splicing can promote transcriptional reprogramming and alter hepatic lipid handling and ketogenic capacity.


Figure 2 Visual predictive check plots. The final disease-drug-trial models characterizing longitudinal normalized weekly binge episodes in patients receiving topiramate and placebo (N = 500 simulations) (a) and binge day frequency (b). Solid and dashed lines represent the observed and model-simulated 5th, 50th, and 95th percentiles. Colored bands demonstrate the 95% confidence interval for the simulated 5th, 50th, and 95th percentiles.

VPC plots for the disease-drug-trial model are shown in Figure 2. Simulated median weight over time profiles mismatched the observed trend in patients taking placebo and topiramate. Model evaluation using QPCs demonstrated the capability of the developed diseasedrug-trial model in predicting key clinical outcomes.


Figure 2. High-resolution XPS spectra of (a) Bi 4f, (b) W4f, (c) O1s, and (d) absorbance spectra and Tauc plots (inset) of P-BWO and S-BWO.

As shown in Figure 2a, the two spin-orbit doublets with peaks emerging at 159.07 and 164.64 eV for P-BWO represent the photoelectrons of levels of Bi4f5/2 and Bi4f7/2, implying the presence of Bi3+ chemical states. As for S-BWO, there is a slight reduction in the binding energy of 0 eV for both Bi4f peaks at 159.07 and 164.37 eV due to the introduction of the S element into the crystal structure. In regard to the W4f spectrum for P-BWO as rendered in Figure 2b, the doublet peaks can be deconvoluted into binding energy of 35.41 and 37.73 eV, which are assigned to the W4f7/2 and W4f5/2 in the W6+ oxidation state of P-BWO. There is a 0.2 eV reduction in binding energy for both peaks of S-BWO, indicating that the chemical environment is altered with the introduction of the S element. As for the O1s core spectrum, the deconvoluted peaks at 530.33 and 531.40 eV for P-BWO are attributed to lattice oxygen and surface chemisorbed oxygen, respectively. The binding energy for both peaks decreased by approximately 0.23 eV for S-BWO, as shown in Figure 2c. The optical properties of both P-BWO and S-BWO were studied using UV/Vis diffuse reflectance spectroscopy. As shown in Figure 2d, the absorption edge of S-BWO exhibits an obvious red-shift compared to that of P-BWO, indicating an increased absorption in the visible light region with the introduction of S.


Figure 2 EA improves liver steatosis and liver function in mice. (A) H&E staining of liver tissue (scale bar = 200 µm). The red arrows indicate typical hepatic inflammation. (B) Liver tissue stained with Oil Red O (scale bar = 200 µm). (C) Morphology of liver. (D) Hepatic TG. (E) Hepatic TC. (F) Serum ALT. (G) Serum AST. n = 6, values are mean ± SEM; *** p < 0.001 (Control vs Model); ## p < 0.01 (Model vs EA); ΔΔ p < 0.01 (EA vs SEA).

HE and Oil Red O staining revealed pronounced lipid deposition in the liver of mice, accompanied by disorganized hepatocyte structure, varying sizes of droplets, and alterations in the MOD group. Notably, the MOD group exhibited significantly weaker abnormal lipid accumulation in the liver compared to the CON group, the group treated with EA had a significant increase in hepatic lipid accumulation, as depicted in Figure 2A-C. In accordance with the Elisa results, the hepatic concentrations of TG and TC in mice from the MOD group exhibited a statistically significant elevation compared to those from the CON group. The addition of EA resulted in a considerable decrease in hepatic levels of TG and TC, as seen in Figure 2D and E. Furthermore, the MOD group exhibited a notable elevation in blood levels of ALT and AST in comparison to the CON group. After the treatment, there was a notable decrease in the serum levels of ALT and AST (Figure 2F and G).


Figure 3. Activation of nuclear factor (NF)-κB signaling by bone morphogenetic protein (BMP)4 stimulation in intraepithelial lymphocytes (IELs). Flow cytometry determined the expression of the BMP type I receptor and phosphorylated NF-κB in IELs in culture. following treatment with BMP4 for 6 h, the expression of the BmP type i receptor and phosphorylated nf-κB was significantly increased compared with that in the control group. noggin partially decreased the expression of the BMP type I receptor, as well as NF-κB transcriptional activity. The results are representative of three independent experiments. P < 0.05.

Exogenous BMP4 regulates the NF-κB signaling pathway in IELs. the primary BmP receptors include two type i receptors (BmPria and BmPriB) and one type II receptor (BMPRII). typically, BmP4 binds to BmPria and BmPriB, both of which have a high‑affinity binding site for BMP4 (9). To determine whether the activation of nf-κB signaling in iels involves iec-derived BmP4, iels from the small intestines of mice were isolated and cultured. it was observed that the protein levels of BmPria, BmPriB and phosphorylated nf-κB were highly upregulated in iels that were cultured in the presence of BmP4 (30 ng/ml), as assessed by flow cytometry (BMPRIA,3.88±0.56%; BMPRIB, 10.00±1.42%; and p-nf-κB, 11.33±1.45%). However, this effect was abolished by the BmP-specific antagonist NOGGIN (30 ng/ml), as shown in Fig. 3. Therefore, it is hypothesized that I/R induces the activation of NF‑κB signaling in iels, possibly through IEC‑derived BMP4.


Figure 4. Transgene expression and influence on tumor growth inhibition of polymer coated adenovirus (PC-Ad) with focused ultrasound (US) in the presence of SonoVue microbubbles (SV). A) Luciferase transgene expression in tumors of mice injected intravenously with PC-Ad and treated simultaneously with US or not, as quantified using IVIS for the first 20 days after injection. Improved luciferase expression in US-treated tumors indicates increased replication and spread. n = 8. Standard deviation is shown by bars. Two-sided t test performed at each time-point. At 1, 2, 3, and 7 days, P =.007, .004, .03, and .004, respectively. B) Staining of tumor sections with anti-CD31 (red) or anti-hexon (green) to determine the influence of US on intratumoral penetration of PC-Ad. Scale bar = 20 µm. Staining of Ad away from endothelial cells suggests improved penetration into tumors. C) Tumor growth in mice treated with phosphate-buffered saline (PBS) (black line), focused ultrasound and SV but no PC-Ad (gray line), PC-Ad with SV without focused ultrasound (blue line), or PC-Ad with SV with ultrasound (green line), as assessed by daily measurement with calipers. n = 8 mice. Mean values ± 95% confidence intervals are shown. Analysis of variance with Tukey correction comparing all groups post-test was used. D) Kaplan–Meier survival curve of mice treated in (C), in accordance with animal welfare legislation, mice were killed when tumor size reached a predefined limit of 700mm³. Log-rank test was performed. Results are typical of two independent experiments. All statistical tests were two-sided.

Mice bearing one ZR-75-1 tumor were injected intravenously with PC-Ad + SV, with or without the application of ultrasound. The replication and lytic spread of Ad was tracked by measurement of luciferase and daily tumor sizing. After 20 hours, luciferase expression in tumors of ultrasound-treated mice exceeded the level in non-ultrasound-treated mice by threefold (P = .007). This enhancement level may be below the level seen in some mice in our previous studies using nonmodified Ad (31), but its dramatically reduced variability provides a substantial benefit. Furthermore, it is notable that this differential increased to 20-fold by 2 days (P = .004) and 30-fold by 3 days (4×10^6 light units; SD = 3×10^6 for PC-Ad+SV+ultrasound group vs 1.3×10^5; SD = 1×10^5 for PC-Ad+SV group; P = .03) (Figure 4A). The possibility that improved intratumoral distribution permits small increases in uptake to translate into dramatic increases in infection was confirmed using fluorescence microscopy of tumor sections (Figure 4B; Supplementary Figure 3F, available online). Tumor growth was statistically significantly accelerated with PC-Ad + SV + ultrasound compared with all other treatment groups. This was the case if mean tumor sizes at day 42, PC-Ad + SV + ultrasound vs PC-Ad + SV (P = .02) (Figure 4C), or mean areas under the curves of tumor growth were compared (PC-Ad + SV + ultrasound; mean = 12 710 mm^3; SD = 3177 vs PC-Ad + SV; 18 318 mm^3; SD = 4665; P = .04). Notably, higher luciferase expression was associated with greater retardation of tumor growth and survival extension. Levels of 2×10^8 to 1×10^9 photons/sec/cm^2 were associated with almost complete growth retardation. Kaplan-Meier analysis demonstrated median days survival values of 22 for PBS, 21 for SV + ultrasound, 26 for PC-Ad + SV = 26, and 33 for PC-Ad + SV + ultrasound (Figure 4D).


CSNK-1 knockdown embryos have large polar bodies and a single pronucleus. (A) DIC images from z-stacks through control vs. csnk-1(RNAi) dissected embryos from three different strains (N2, FM99, and FM135). Images were acquired between pronuclear migration and pronuclear breakdown. Arrows mark each visible polar body. Scale bar, 10 μm. (B) Graph of polar body area in N2, FM99, and FM135 control vs. csnk-1(RNAi) embryos. Two-dimensional area was measured in the z-stack where the polar body was at its largest size. Statistical analysis was by two-tailed Student’s t test. ***, p < 0.001. (C) Representative images of a control N2 embryo and a csnk-1(RNAi) embryo at midfocal plane after pronuclear migration. Scale bar, 10 μm. (D) Graph of the number of pronuclei present in the embryo after female meiosis in control vs. csnk-1(RNAi) embryos. The fraction of csnk-1(RNAi) embryos with a single pronucleus in the three strain backgrounds tested was not significantly different (Fisher’s exact test p = 0.25).

Whereas two small polar bodies were observed in control embryos, csnk-1(RNAi) embryos had very small polar bodies (Figure 1A). Polar body size was measured in DIC z-stacks of embryos between pronuclear migration and pronuclear centration as the two-dimensional area of each polar body in the focal plane with the largest area (Figure 1B). In contrast, 12 out of 45 csnk-1(RNAi) embryos had a single pronucleus (Figure 1C and 1D), which is clearly visible in csnk-1(RNAi) embryos (Figure 1A and 1C).


GRASP55 deletion has minor effects on the Golgi structure. (A) Western blots of Golgi proteins in GRASP55 knockout HeLa cells. Wild-type and representative GRASP55 knockout clones from three separate sgRNAs (T1, T2, and T3) were lysed and blotted for GRASP55/65, Golgin-45, and GM130. (B) Quantification of A for the relative levels of GRASP65, Golgin-45, and GM130 in GRASP55 knockout cells. Error bars represent SEM. (C) Immunofluorescence of GRASP55 knockout clones stained for GM130 and TGN46. The lower three rows are increased magnifications of the Golgi in a single cell. Scale bars are 10 µm. (D) Colocalization of GM130 and TGN46 quantified by the Pearson’s correlation coefficient of z-stacks from GRASP55 knockout clones from C. Error bars represent SEM. (E) Quantification of Golgi fragmentation in GRASP55 knockout clones in C. Blinded determination of the Golgi morphology of 300 cells from each sample was quantified across three biological replicates. Error bars represent SEM. A Student’s t test was performed to determine statistical significance. *p < 0.05.

GRASP55 deletion also resulted in a significant increase of Golgin-45 in HeLa cells, while GM130 protein levels remained unchanged in both cell lines (Figure 2A and 2B and Supplemental Figure S3A and S3B). Deletion of GRASP55 caused a minor but significant decrease in the level of Golgi fragmentation in both HeLa and HEK293 cells, as assessed by immunofluorescence microscopy for GM130 and TGN46 (Figure 2C–2E and Supplemental Figure S3C–S3E). However, colocalization of GM130 and TGN46, as measured by Pearson’s correlation coefficient, remained changed in HeLa cells. Western blot analysis of GRASP55 knockout clones demonstrated that GRASP55 depletion was effective, as no GRASP55 signal was detected (Figure 2A and Supplemental Figure S3A). Knockout of GRASP55 significantly increased the level of GRASP65 in HEK293 cells (Supplemental Figure S3A and S3B), although this effect was not as obvious in HeLa cells (Figure 2A and 2B).


Fig. 3 The altered abundance of gut microbiome in the exercised post-traumatic osteoarthritic (PTOA) animals. a) A Venn diagram was generated to compare operational taxonomic units (OTUs) among three groups and to depict OTUs that were unique to the three groups. b) Taxonomic profiles of the fecal bacteria from 16S ribosomal ribonucleic acid (rRNA) gene sequencing show the relative abundance of the top 10 phyla and the top 10 genera of fecal bacteria presented in the Sham/sedentary (Sed), PTOA/Sed, and PTOA/treadmill-walking (TW) groups. Changes in the c) two bacterial phyla and d) five genera in the gut microbiome composition of three groups. p < 0.05, p < 0.01, PTOA/Sed or PTOA/TW versus Sham/Sed, one-way analysis of variance (ANOVA); #p < 0.05, ##p < 0.01, PTOA/Sed versus PTOA/TW, one-way ANOVA; ns, not significant. Data are presented as the mean and standard deviation, n = 6.

The core OTUs comprised 6.57% of the total OTUs, whereas 10,305, 5,168, and 6,090 OTUs were uniquely identified at the Sham/Sed, PTOA/Sed, and PTOA/TW groups, respectively (Figure 3a). The OTUs were classified into 36 bacterial phyla and 432 genera. According to the number of reads in the sequence, the top 10 phyla and genera in relative abundance of the fecal microbiome are displayed in Figure 3b and Supplementary Figure c. Some differences identified at the levels of phylum and genus of fecal microbiome among the three groups are displayed in Figure 3c. The relative abundance of the phylum TM7 was consistently lower in both PTOA/Sed and PTOA/TW groups compared to the Sham/Sed group. The phylum Fusobacteria, which was robustly abundant in the gut of the PTOA rats without exercise, was nearly absent in the Sham/Sed and PTOA/TW groups. In this context, phylum TM7 and Fusobacteria were identified as the PTOA-relevant phyla and exercise decreased the abundance of Fusobacteria. At the genus level, the relative abundance of Lactobacillus, Turicibacter, Adlercreutzia, and Cetobacterium was increased in the PTOA/Sed group compared to Sham/Sed group, while the PTOA-induced Adlercreutzia and Cetobacterium were reduced by exercise (Figure 3d).


Figure 5. Effect of HA on OC-like cell adhesion to FN. (A) Assessment of the relative avidity of cell adhesion onto FN in the presence of HA by varying the centrifugal force (45 g, 180 g, and 400 g) applied to dislodge bound cells. Cells were allowed to adhere to FN coated at 5 µg/ml. (B) Adhesion to FN in the presence of HA or the 4B4 function-blocking anti-β1 mAb. (C) Assessment of the relative avidity of cell adhesion to FN of cells maintained for 24 h in the presence of soluble HA by varying the centrifugal force (45 g, 180 g, and 400 g) applied to dislodge bound cells. (D) Phase-contrast (p.c.) morphology and CD44/actin staining of adherent OC-like FLG 29.1 cells to FN-coated slides (20 µg/ml) at 20 µg/ml in the absence or presence of 1.0 mg/ml HA. Bars, 100 µm.

Soluble HA (Figure 5B). Another possibility could be that the addition of soluble HA could affect, via an inside-out mechanism mediated by the cytoplasmic domain of CD44, the affinity/avidity of integrin receptors for their cognate ligands. Thus, additional adhesion experiments were performed with OC-like FLG 29.1 cells grown in the presence of soluble HA for 24 h and then assayed with CAFCA at different detachment forces (45 g, 180 g, and 400 g) in the absence of HA. Again, a significant difference was ated when compared with control cells grown in the absence of HA (Figure 5C). Consistently, the presence of soluble HA affected neither the expression levels of α5β1 and αvβ1 integrins on OC-like FLG 29.1, as evaluated by flow cytometry (unpublished data), nor the morphology of cells attached to FN, nor the even cell membrane distribution of CD44 molecules (Figure 5D).


Inhibition by NPPB, furosemide, and sucrose of the sorbitol-induced hemolysis. (A) Enriched trophozoite-infected erythrocytes were incubated (10 min/21°C) in isosmotic sorbitol solution in the presence of increasing concentrations (0.01–30 µM) of NPPB (open triangles; SE, n = 12) or furosemide (closed triangles; SE, n = 9). Incubation was stopped by centrifugation and hemolysis was indicated by hemoglobin in the supernatant. In the insert on the right are the scanned images of supernatants from an individual experiment. The hemoglobin concentration of the supernatant for each condition (NPPB and furosemide) was determined photometrically (546 nm). Data are expressed as the percentage of the 30 µM NPPB inhibited fraction (maximal inhibition ≈ 86 ± 2% of total hemolysis, left). (B) Substrate competition of the infection-induced sorbitol hemolysis. Hemolysis (10 min/21°C) was assessed in solutions where different quantities of sorbitol (0–120 mosM) have been isosmotically replaced by sucrose (circles) or NaCl (squares). Mean normalized hemolysis (± SE; n = 8; left) and imaged supernatants of an individual experiment (right) are shown as in A. (C) Uptake of [¹⁴C] sorbitol into P. falciparum–infected human erythrocytes incubated in isosmotic sorbitol solution. The uptake of radiolabeled sorbitol was measured after 5, 30, 60, 90, and 120 s of incubation. Individual data of three experimental series were expressed as a percentage of the respective 2 min values and averaged (means ± SE; n = 4–12). (D) Inhibition of [¹⁴C]sorbitol uptake in isosmotic sorbitol by sucrose. Mean uptake (± SE; n = 9–10) of [¹⁴C]sorbitol (30 s values minus the 5 s blank value) in isosmotic sorbitol solution (control) or in sorbitol solutions where 20 and 60 mM of sorbitol were replaced by 20 and 60 mM of sucrose or 10 and 30 mM of NaCl, respectively (uptake conditions: 3.7 × 10⁴ Bq/ml [¹⁴C]sorbitol, 2.5% final hematocrit, 90% parasitemia). * and # correspond to P < 0.01 and 0.05, respectively, ANOVA.

Finally, the NPPB and furosemide inhibition of the hemolysis in isosmotic sorbitol solution was compared with those of both conductances (Figure 7A and B). NPPB and furosemide inhibited the sorbitol hemolysis of infected erythrocytes in a dose-dependent manner (Figure 7A) with IC₅₀s (~1 µM for NPPB and ~10 µM for furosemide; Figure 7A) similar to those for the outward rectifying lactate current (Figure 3D). In addition, the effect of sucrose on the infection-induced hemolysis was determined by isosmotic substitution of sorbitol with increasing concentrations of sucrose and for control of NaCl (Figure 7B). Replacement by sucrose but not by NaCl inhibited hemolysis of trophozoite-infected cells with an IC₅₀ of ~10 mM, indicating (a) competition between sorbitol and sucrose within the permeability pore and (b) low permeability of sucrose. To further test for the inhibitory effect of sucrose on the sorbitol pathway of infected erythrocytes, isotopic flux measurements of [¹⁴C]sorbitol were performed under the experimental conditions used for the hemolysis experiments. Figure 7C shows the time course of the [¹⁴C]sorbitol uptake in isoosmotic sorbitol solution. Within 2 min of incubation the uptake apparently saturated. Incubation (30 s) of the infected cells in the presence of 20 or 60 mM sucrose (replacing the same amount of sorbitol) significantly decreased the uptake of [¹⁴C]sorbitol as illustrated in Figure 7D. In contrast, substitution of 20 and 60 mM sorbitol by 10 and 30 mM NaCl did not significantly decrease [¹⁴C]sorbitol uptake, respectively (Figure 7D).


Ccdc11 is a component of centriolar satellites. (A) Immunoblot of lysates from tetracycline-inducible GFP-Ccdc11–expressing RPE-1 stable cell line (RPE::GFP-Ccdc11) probed with anti-Ccdc11 antibody. Numbers on the left indicate molecular mass of markers in kilodaltons. (B) Localization of endogenous Ccdc11 throughout the cell cycle. Asynchronously growing MEF cells were fixed and stained for Ccdc11 (green), centrin to identify centrioles (red), acetylated tubulin to mark cilia (red), and DNA (4′,6-diamidino-2-phenylindole [DAPI]; blue). The centriolar satellite distribution of Ccdc11 is conserved through G1, S phase, G2, and mitosis. After serum starvation to induce ciliogenesis, the satellite pool of Ccdc11 is lost, and the protein is enriched at the transition zone (distal end of each centriole, arrowheads). Insets, magnified images of the centrosome region. Scale bar, 5 μm. (C) RPE::GFP-Ccdc11 cells immunostained with antibodies against GFP (green), PCM-1 (red), or Cep290 (red), as well as centrin (magenta) or acetylated tubulin (light blue) to mark centrioles and cilia, respectively. Ccdc11 colocalizes with PCM-1 and Cep290 at satellites in G1 and with Cep290 at the transition zone in G0. Scale bar, 5 μm. (D, E) RPE-1 cells were treated with nocodazole for 1 h to depolymerize microtubules and then allowed to recover for up to 30 min. Samples were fixed at the indicated times and stained for Ccdc11 (green), centrioles (centrin, magenta), microtubules (α-tubulin, red), and DNA (DAPI, blue). Ccdc11 localization to centriolar satellites is microtubule dependent, but the centriolar pool of Ccdc11 does not require microtubules. Scale bar, 10 μm. Arrowhead indicates the satellite pool of Ccdc11. We scored 300 cells for each sample, from three independent experiments; **p < 0.05. (F) Ccdc11 interacts with satellite proteins PCM-1 and Cep290. Immunoprecipitation was performed on extracts from RPE::GFP-Ccdc11 cells using anti-GFP antibody or control IgG and then probed for GFP and endogenous PCM-1, Cep290, and γ-tubulin (negative control).

Ccdc11 is a protein of 514 amino acids with a predicted molecular weight of ~75 kDa containing three putative coiled-coil domains. It is conserved in organisms containing centrioles/basal bodies and cilia ranging from humans to Chlamydomonas (Supplemental Figure S1, A–C). To characterize the localization and function of Ccdc11 at the cellular level, we generated polyclonal antibodies directed against human Ccdc11. The antibody recognized a protein of expected size in immunoblots of human telomerase–immortalized retinal pigment epithelial cells (hTERT-RPE-1; here after referred to as RPE-1; Figure 1A). The antibody also recognized the green fluorescent protein (GFP)–tagged fusion protein in RPE-1 cells stably expressing tetracycline-inducible GFP-Ccdc11 (RPE::GFP-Ccdc11; Figure 1A). Next we investigated the localization of endogenous Ccdc11 at various stages of the cell division cycle. Ccdc11 was present on both centrioles in G1 cells and was also distributed in small aggregates that surrounded the two centrioles (Figure 1B). This localization pattern is highly reminiscent of centriolar satellites, which are 70- to 100-nm cytoplasmic granules that concentrate around the centrioles/centrosome and are involved in trafficking of proteins to and from the centrosome (Kubo et al., 1999; Dammermann and Merdes, 2002).


Figure 2. Efficient entry of HIV-1 into DCs but not small T cells. The results are representative of 2 or more experiments in each case. (A) Early vs. late stages of reverse transcription in blood-derived DCs. IIIB was added for 90 min at 37°C to DCs, purified T cells (T), and T blasts (TBl), washed, and cultured 0, 4, 18, or 36 h. Early and late (R/U5 and LTR/gag primers) transcripts were amplified by PCR and compared to graded doses of ACH-2 cells. (B) Several isolates of HIV-1 efficiently enter blood-derived DCs. Graded doses of the denoted isolates (titred by the AIDS Resources Program) were added to DCs for 90 min, washed, and cultured 4 h before amplifying R/U5 DNA.(C) Detection of early R/U5 transcripts 3 h after adding Ba-L and IIIB isolates to skin leukocytes, either bulk skin cells (SC), FACS-purified DCs and T cells, or a 1:2 DC/T mixture. (D and E) Evidence that late reverse transcripts in HIV-1 pulsed DCs represent viral entry and not simply binding to skin leukocytes (D) or blood derived DCs (E). R/U5 sequences are infrequent if virus was bound for 4 h on ice, and was trypsin insensitive (0.25%, GIBCO for 10 min at 37°C after 4 cell washes in phosphate saline) if virus was offered for 4 h at 37°C. (D) Kinetics of R/U5 sequence formation with Ba-L or IIIB in skin cells (SC) and T blasts. The 0 h time point was carried out 90 min after adding virus at 37°C. DNA was also analyzed 1–8 h after the virus pulse, washing, and culture.

At time 0, i.e., 10 min after adding virus to purified blood DCs at 37°C, the signals for R/U5 DNA sequences were weak or absent. HIV-1 DNA then decreased steadily for 8 h to reach a plateau (Fig. 2 A) for at least 1–3 d (not shown). Only the early products of reverse transcription were abundant. At the dose of virus we employed, and relative to a standard curve derived from the ACH-2 cell line that expresses 1 copy of proviral DNA/cell (15), we observed 103–104 copies of R/U5 DNA in 100,000 DCs and .102 copies of LTR/gag sequences (Fig. 2 A). This signal is very high considering that we were only applied virus at ,0.1 MOI. R/U5 DNA was weak or not detected with omparable numbers of purified T cells (Fig. 2 A). T blasts were readily infected, as both R/U5 and LTR/gag containing sequences were found. The amount of these sequences increased rapidly in culture, because infection was productive in T blasts (Fig. 2 A). Several different HIV-1 isolates entered and began reverse transcription in DCs (Fig. 2 B). Using comparable numbers of infectious units, the interaction of Ba-L, SF162 and IIIB was particularly efficient. Efficient entry of these and other primary isolates was also noted in skin DCs, more than skin T cells, but only with probes for R/U5 sequences (Fig. 2 C). Given the evidence that HIV-1 virions can contain short reverse transcripts (22, 23), we were concerned that the R/U5 sequences represented intraviron-DNA that had bound but not entered the DCs. However, entry seemed more likely for the following reasons. The R/U5 signal was strong when virus was added on ice for 4 h (Fig. 2, D and E), even though virus does bind in the cold (23–25). The R/U5 signals were weak if the cells were examined just after the 90-min virus pulse at 37°C, but increased progressively for 8 h after washing away nonbound virus (Figs. 2, A and F). In contrast, virus binding can be detected in just 3-5 min at this temperature (23–25). The R/U5 signals that developed after 4 h at 37°C also were insensitive to trypsin (Fig. 2, D and E), which releases surface-bound virus (23).


Figure 2 Plk3 and procaspase-8 interact. (A) HEK 293T cells were co-transfected with GFP-Plk3 or GFP-Rip3 and Flag-procaspase-8. GFP-Rip3 was used as a positive control for the co-IP with procaspase-8. Precipitates were immunoblotted for Flag and GFP. (B) The binding ability of GST-fused procaspase-8 was analyzed in pull-down assays. GST-procaspase-8 or GST alone (as a control) wasincubated with HeLa cell lysates. The pull-down was analyzed by immunoblotting for Plk3 and GST. (C) Schematic representation of recombinant GST-fused full-length Plk3 (GST-Plk3-WT) and truncated forms of Plk3 (GST-Plk3-NT1, -NT3 and -CT1) used in pull-down assays. (D) GST-Plk3-WT or Plk3 subdomains were incubated with HeLa cell lysates for pull-down assays. The immunoblot analysis was performed using caspase-8- and GST-antibodies. (E) HeLa cells were treated with 50 ng/ml nocodazole (Noc) for 16 h to enrich for mitotic cells. Endogenous caspase-8 was immunoprecipitated from cell lysates. Normal mouse IgG was used as a control. The immunoprecipitates and the lysates were immunoblotted for caspase-8, Plk1, Cdk1, Cyclin B1, Plk3 and Aurora B. (F) HeLa cells were treated with 50 ng/mL CD95L and CHX for 0 h and 2 h. Endogenous caspase-8 was immunoprecipitated from cell lysates with anti-caspase-8 p18 goat antibody. Normal goat IgG (Santa Cruz) was used as a control. The immunoprecipitates and the lysates were immunoblotted for Plk3, caspase-8, cleaved caspase-8 and p10. (G) The interaction of Plk3 and procaspase-8 (Casp8) was monitored via in situ PLA. HeLa cells were labeled with the anti-Plk3 (Abcam, rabbit) and anti-Casp8 (mouse) (left panel) or anti-Plk3 (BD, mouse) and anti-Casp8 (rabbit) (right panel) antibodies. Single antibody staining (Plk3 or Casp8) was used as a control. Scale bar: 10 µm. Each bar represents the mean value ± SD (n = 3). The differences between single and double antibody staining were statistically significant by Student’s t-test (lower panels) (*P ≤ 0.05; **P ≤ 0.005).

Plks are characterized by their PBD, which mediates protein interactions and provides target specificity. Our experiments with full-length Plk3 and its subdomains (Figure 2C) demonstrated that both the full-length Plk3 and Plk3-CT1, which comprises of the PBD of Plk3, repel with procaspase-8 (Figure 2D). In an IP-analysis of endogenous protein we confirmed the association of procaspase-8 with Plk1 and Cdk1/cyclin B1 in mitotic cells [31, 32] and corroborated the interaction of caspase-8 and Plk3, which is independent of mitosis (Figure 2E). Stimulation of CD95 receptor induced processing of procaspase-8 with increasing levels of p18 and p10 (Figure 2F). Simultaneous IP of procaspase-8, whose levels decline upon CD95 stimulation, showed decreasing amounts of co-precipitated Plk3 (Figure 2F, right panel). The quantification of the PLA using Plk3 antibodies targeting the N-terminal (Abcam) or the interdomain of Plk3 (BD) refuted the close proximity of both endogenous proteins in HeLa cells (Figure 2G, upper and lower panel)


Figure 1. TROY associates with PDZ-RhoGEF (PRG). (A) 293 cells were co-transfected with HA-tagged TROY plasmid and Myc-tagged PDZ-RhoGEF plasmid, and the lysates were immunoprecipitated with an anti-Myc antibody or mouse IgG. The immunoprecipitates were analyzed for the presence of PDZ-RhoGEF by immunoblot analysis. The expression level of Myc-tagged PDZ-RhoGEF and HA-tagged TROY in the cells is shown by immunoblotting of whole cell lysates (WCL). (B-D) The lysates of T98G, GBM10, and GBM43 cells were immunoprecipitated with an anti-TROY antibody or rabbit IgG. The immunoprecipitates were analyzed for the presence of PDZ-RhoGEF by immunoblot analysis. The expression level of PDZ-RhoGEF and TROY in the cells is shown by immunoblotting of WCL. (E) TROY does not co-immunoprecipitate with LARG in 293 cells. 293 cells were transfected with HA-tagged LARG or Myc-tagged TROY, or co-transfected with HA-tagged LARG and Myc-tagged TROY. Twenty-four hours after transfection, cells were lysed and immunoprecipitated with anti-Myc antibody. Immunoprecipitates or WCL were immunoblotted with the indicated antibodies. (F) Colocalization of TROY and PDZ-RhoGEF in T98G, GBM10, and GBM43 cells. Cells were stained and examined by confocal microscopy. TROY was visualized with Alexa Fluor 546–conjugated antibody, and PDZ-RhoGEF was visualized with Alexa Fluor 488–conjugated antibody. The colocalization of TROY (red) with PDZ-RhoGEF (green) appears as a yellow color in the merged image. Cells were counterstained with DAPI (blue) (bars = 10 μm). (G) 293 cells were co-transfected with AU1-tagged TROY, Myc-tagged PRG, and HA-tagged Pyk2, and 24 hours later, cells were lysed and immunoprecipitated with goat anti-Myc or goat anti-AU1 antibodies or goat IgG. Immunoprecipitates or WCL were immunoblotted (IB) with the indicated antibodies. (H) Left panel, 293 cells were transfected with HA-tagged LARG and/or FLAG-tagged Pyk2. Twenty-four hours later, the cells were lysed and immunoprecipitated with anti-FLAG antibody or mouse IgG. Immunoprecipitates or WCL were IB with anti-FLAG or anti-HA antibodies. Right panel, 293 cells were co-transfected with Myc-tagged PRG and FLAG-tagged WT Pyk2 or kinase-deficient (KD) Pyk2 for 24 hours. Cells were lysed and immunoprecipitated with anti-Myc antibody. Immunoprecipitates or WCL were IB with the indicated antibodies.

To determine possible mechanisms through which TROY induces GBM cell invasion, we performed immunoprecipitation of TROY from T98G glioma cells overexpressing TROY and analyzed the precipitates with MALDI-TOF and MS/MS to identify proteins that are capable of interacting with TROY and potentially mediating TROY signaling [13]. Several proteins were identified in the TROY immunoprecipitates, among which PDZ-RhoGEF was of particular interest because it has previously been reported to be involved in cellular motility [30,31]. To confirm the interaction between TROY and PDZ-RhoGEF, 293 cells co-transfected with HA-tagged TROY and Myc-tagged PDZ-RhoGEF were immunoprecipitated with an anti-Myc antibody, and the precipitates were immunoblotted with anti-HA and anti-Myc antibodies. Immunoblotting demonstrated that HA-tagged TROY co-immunoprecipitated with Myc-tagged PDZ-RhoGEF, refuting an interaction between TROY and PDZ-RhoGEF (Figure 1A). Endogenous PDZ-RhoGEF was found to co-immunoprecipitate with endogenous TROY in T98G glioma cells as well as primary GBM patient-derived xenografts GBM10 and GBM43 cells (Figure 1, B-D). The expression pattern of endogenous TROY and TROY interactants in the different cell lines is shown in Supplementary Figure 1. To explore the specificity of the interaction between TROY and PDZ-RhoGEF, we examined whether TROY could interact with LARG, a closely related GEF belonging to the same RhoGEF subfamily as PDZ-RhoGEF. 293 cells were co-transfected with HA-tagged LARG and/or Myc-tagged TROY. Immunoprecipitation of the transfected cell lysates with an anti-Myc antibody followed by immunoblotting of the precipitates with the anti-HA antibody showed that LARG was not present in the TROY-Myc immunoprecipitates, suggesting a specific interaction of TROY with PDZ-RhoGEF (Figure 1E). To further confirm the interaction, we examined the colocalization of TROY and PDZ-RhoGEF by immunofluorescence analysis. T98G, GBM10, and GBM43 cells were stained with anti-TROY and anti–PDZ-RhoGEF antibodies and examined by confocal microscopy (Figure 1F). Consistent with previous results [13], TROY staining was predominately near the cell periphery and enriched in lamellipodia, and the merged images contradicted colocalization of TROY and PDZ-RhoGEF.


The image was divided ino a 3-row by 3-column equal-area grid, resulting in 9 blocks.
The numbering goes left to right， top to bottom:
Block 1 = first row,left
Block 2=first row, center
Block 3 =first row, right
Block 4 = second row, left
Block 5 = second row, center
Block 6= second row, right
Block 7= third row, left
Block 8= third row, center
Block 9= third row, right


The image was divided into a 3-row by 3-column equal-area grid, resulting in 9 blocks.
The numbering goes left to right, top to bottom:
Block 1 = first row, left
Block 2 = first row, center
Block 3 = first row, right
Block 4 = second row, left
Block 5 = second row, center
Block 6 = second row, right
Block 7 = third row, left
Block 8 = third row, center
Block 9 = third row, right