Figure 4. Fas signaling is activated by redistribution of Fas into lipid rafts in cells overexpressing 3OST-5. (A) Western blot analysis of raft and non-raft fractions, using anti-Flotillin-1 (raft), anti-transferrin receptor (non-raft), and anti-Fas antibodies, of mESCs at 2 days after transfection with the 3OST-5 expression construct (upper two panels) or mESCs at 6 days after LIF withdrawal (lower two panels). At least two independent experiments were performed. Representative results are shown. (B) Western blot analysis, using antibodies against uncleaved and cleaved caspase-8, of mESCs at 2 days after transfection with the 3OST-5 expression construct (left and middle right panels) or mESCs at 8 days after LIF withdrawal (middle left and right panels). The histograms show mean densitometric readings 6 SD after normalization against HS4C3-Binding Epitope Contributes to Fas Signaling control cells (left and middle right panels) or mESCs cultured in the presence of LIF (middle left and right panels) (arbitrary value = 1). Three independent experiments were performed. *, P,0.01; **, P,0.05. (C) FACS analysis, using the anti-Fas antibody, of mESCs at 2 days after transfection with the Fas expression construct. In the left panel, a histogram shows a representative result of the FACS analysis (black line, control cells; red line, cells overexpressing Fas). The gray line shows the result obtained for cells that were not treated with the primary antibody. In the right panel, the values shown are means 6 SD after normalization against control cells (arbitrary value = 1). Three indepen- dent experiments were performed. *, P,0.01. (D) Self-renewal assay in cells overexpressing Fas. The proportion of AP-positive colonies is shown. The values shown are the mean 6 SD. Two days after transfection, mESCs were replated in ESC medium with or without LIF. *, P,0.01. (E) Mutations and truncations of the recombinant Fas ectodomain. (F) and (G) Overlay assay using the GST-fused recombinant Fas ectodomain. F-1 and G-1 show a western blot using the HS4C3 antibody. The single asterisk (*) shows the effect of the increase in the HS4C3-binding epitope on several core proteins in cells overexpressing 3OST-5. F-2 and G-2,-4 show the overlay assay using the Fas ectodomain (F-2, aa 19–168) or fragments of the Fas ectodomain (G-2, aa 19–38; G-4, aa 39–168). F-3 and G-3,-5 show the overlay assay using the Fas ectodomain (F-3, aa 19–168) or fragments of the Fas ectodomain (G-3, aa 19–38; G-5, aa 39–168) pre-mixed with HS4C3 antibody. F-4 shows the overlay assay using the mutated Fas ectodomain (aa 19–168). The double asterisk (**) shows increased binding of the Fas ectodomain in cells overexpressing 3OST-5. b-actin was used as a loading control for each sample (F-5 and G-6). mESCs at 2 days after transfection with the 3OST-5 expression construct were used for each analysis. Two independent experiments were performed. Representative results are shown. GST, glutathione S-transferase. doi:10.1371/journal.pone.0043440.g004 Differentiation of mESCs Induced by Overexpression of the HS4C3-Binding Epitope is Inhibited by Blockage of Fas Signaling To confirm that activation of Fas signaling via overexpression of the HS4C3-binding epitope was involved in mESC differentiation, we investigated whether the reduced self-renewal capability of cells overexpressing 3OST-5 could be rescued by blocking Fas signaling using the peptides Ac-IETD-CHO (IETD) and Ac-DEVD-CHO (DEVD), which block the activity of caspase-8 and caspase-3, respectively. We found that treatment of cells overexpressing 3OST-5 with the inhibitors inhibited caspase-3 activation and rescued the degradation of Nanog protein (Figure 5A and 5B). Next, we compared the morphologies of control cells and cells overexpressing 3OST-5. Control cells had an undifferentiated appearance with a moderately packed morphology (Figure 5C). In contrast, almost all cells overexpressing 3OST-5 had a flattened, differentiated morphology (Figure 5C). Some IETD-treated cells had a similar morphology to undifferentiated mESCs (Figure 5C). Then, we performed a self-renewal assay and counted the AP- positive colonies. Treatment of cells overexpressing 3OST-5 with DEVD or IETD restored the proportion of AP-positive colonies to a level similar to that obtained with control cells (Figure 5D). Furthermore, the level of Oct3/4 and Nanog mRNA in cells overexpressing 3OST-5 was higher after IETD treatment than in untreated cells (Figure 5E). Thus, we demonstrated that activation of Fas signaling via overexpression of the HS4C3-binding epitope induced differentiation in mESCs. As shown in Figure 4B, Fas signaling was activated in mESCs after the induction of differentiation by LIF withdrawal. We expected that blocking Fas signaling would inhibit the induction of differentiation by LIF withdrawal. Indeed, resistance to differentiation caused by activation of Fas signaling was observed in mESCs treated with IETD in the absence of LIF (Figure 5F). These results indicated PLOS ONE | www.plosone.org 9 August 2012 | Volume 7 | Issue 8 | e43440