HS4C3-Binding Epitope Contributes to Fas Signaling Figure 5. Differentiation induced by overexpression of the HS4C3-binding epitope is inhibited by an inhibitor of Fas signaling. (A) and (B) Western blot analysis using antibodies against cleaved caspase-3 and Nanog in the presence or absence of IETD or DEVD, peptides that block caspase-8 and caspase-3, respectively. Representative results are shown. The histograms show mean densitometric readings 6 SD after normalization against cells overexpressing 3OST-5 but not treated with IETD or DEVD (arbitrary value = 1). mESCs were analyzed at 2 days after transfection with the 3OST-5 expression construct. (C) Representative photomicrographs of transfected cells in the presence or absence of IETD. Scale bars, 200 mm. A triple asterisk (***) indicates a high magnification image of the boxed area (Scale bars, 100 mm). mESCs were analyzed at 4 days after transfection with the 3OST-5 expression construct. (D) Self-renewal assay with cells overexpressing 3OST-5 treated with IETD or DEVD. Left panels show photographs of representative colonies. Scale bars, 200 mm. The right panel shows the proportion of AP-positive colonies. The values shown are the mean 6 SD. Two days after transfection, mESCs were replated in ESC medium with LIF. mESCs were cultured with inhibitors throughout the period from transfection to AP staining. (E) Real time PCR analysis of markers of the undifferentiated state. The values shown are means 6 SD after normalization against cells overexpressing 3OST-5 but not treated with IETD (arbitrary value = 1). mESCs were analyzed at 4 days after transfection with the 3OST-5 expression construct. (F) Self-renewal assay after treatment with IETD in the presence or absence of LIF. The ratio of AP-positive colonies is shown. The values shown are the mean 6 S.D. IETD, Ac-IETD-CHO; DEVD, Ac-DEVD-CHO; AP, alkaline phosphatase. *, P,0.01; **, P,0.05. Three independent experiments were performed in each case. doi:10.1371/journal.pone.0043440.g005 that Fas signaling induces the differentiation caused by LIF withdrawal in mESCs. Taken together, rescue experiments that involved blocking Fas signaling demonstrated that the degradation of Nanog protein and induction of differentiation were actually caused by Fas signaling via HS4C3-binding epitope. Decrease in the HS4C3-Binding Epitope Inhibits Differentiation and Apoptosis To examine and confirm the requirement for HS4C3-binding epitope for the differentiation of mESCs, we performed stable and transient knockdown (KD) of 3OST-5 mRNA using RNAi. We designed two constructs that targeted 3OST-5 (3OST-5-1 and 3OST-5-2, which expressed different siRNAs targeting 3OST-5) and one that targeted EGFP as a negative control. The level of 3OST-5 expression was reduced in both stable and transient 3OST- 5 KD cells (Figure 6A and 6J). FACS analysis showed that the HS4C3-binding epitope was decreased in both stable and transient 3OST-5 KD cells (Figure 6B and K). Then, we performed a self- renewal assay with the stable 3OST-5 KD cells. The number of AP-positive colonies did not differ between the stable 3OST-5 KD cells and the control cells in the presence of LIF and serum in clonal density culture (Figure 6C). Furthermore, the expression of markers of the undifferentiated and differentiated states did not change even in the stable 3OST-5 KD cells (Figure S4). These results demonstrated that the reduction in the HS4C3-binding epitope did not affect the self-renewal capability of mESCs. To determine whether down-regulation of the HS4C3-binding epitope affected the potential of mESCs for differentiation, stable 3OST-5 KD cells were induced to form primitive endoderm by LIF withdrawal for 6 days. In the stable 3OST-5 KD cells, the increase in the expression of Gata6 (primitive endoderm marker) that was seen in the control cells was inhibited (Figure 6D). This finding indicated that the HS4C3-binding epitope was necessary for differentiation into primitive endoderm. Next, we investigated in vitro differentiation into embryoid bodies (EBs), which comprise three germ layers: endoderm, mesoderm, and ectoderm. Expres- sion of the HS4C3-binding epitope was increased during EB formation in control cells (Figure 6B). In EBs derived from stable 3OST-5 KD cells, HS4C3-binding epitope was decreased com- pared with that in control EBs (Figure 6B). In turn, the expression of Fgf5 (primitive ectoderm marker), Goosecoid (mesoderm marker), Sox17 (endoderm marker), and Pax6 (ectoderm marker) was decreased by down-regulation of the HS4C3-binding epitope (Figure 6E). Furthermore, Nanog and Oct3/4 were expressed at a higher level in stable 3OST-5 KD cells than in control cells at 4 days after EB formation (Figure 6E). These data demonstrated that differentiation into all three germ layers was inhibited by down- regulation of the HS4C3-binding epitope during EB formation. Then, we examined Fgf4/Erk signaling, which is reported to be a trigger of stem cell differentiation [18], and observed no differences in the level of phosphorylated Erk1/2 between stable 3OST-5 KD cells and control cells after exposure to Fgf4 (Figure 6F). Hence, Fgf4/Erk signaling did not contribute to the reduction of the potential for differentiation in stable 3OST-5 KD cells. Given the result obtained in the present study that the HS4C3-binding epitope contributed to Fas signaling during the differentiation of mESCs into primitive endoderm, we expected that Fas signaling would also function during EB formation. Therefore, we used IETD, a caspase-8 inhibitor, to analyze the role of Fas signaling in EB differentiation. Treatment with IETD throughout EB differentiation led to a reduction in the expression of Fgf5 (primitive ectoderm marker) and Goosecoid (mesoderm marker), which indicated that Fas signaling was necessary for EB differentiation (Figure 6G). The findings demonstrated that Fas signaling via the HS4C3-binding epitope induced normal EB differentiation. In addition, in EBs derived from cells overexpress- ing 3OST-5, expression of the HS4C3-binding epitope, Fgf5, and Goosecoid were increased as compared with EBs derived from control cells (Figure 6H and 6I). Furthermore, as shown in Figure 6L, the population of annexin V-positive cells was increased in control cells after LIF withdrawal for 24 hours, whereas that of annexin V-positive cells did not increase in response to LIF withdrawal in 3OST-5 transient KD cells. These data showed that Fas signaling via the HS4C3-binding epitope was indispensable for the induction of apoptosis and differentiation of mESCs into primitive endoderm and EBs. Fas Colocalizes with HS4C3-Binding Epitope in mESCs We examined the localization of HS4C3-binding epitope and Fas in mESCs during differentiation in response to LIF withdrawal. In the undifferentiated state in the presence of LIF, confocal slices showed that the HS4C3-binding epitope was colocalized with Fas in the intracellular Golgi compartment around the nucleus, not on the cell surface (Figure 7A, indicated by the arrowheads in the upper panels). However, in mESCs induced to differentiate by culture in the absence of LIF for 5 or 7 days, expression of the HS4C3-binding epitope increased, and colocalization of the HS4C3-binding epitope and Fas was observed as dots on the cell surface (Figure 7A, indicated by the arrowheads in the middle and lower panels). These images were consistent with the increase in HS4C3-binding epitope and Fas on the surface of differentiated cells that was shown by the FACS analysis in Figure 3A, and supported the redistribution of Fas into lipid rafts that was indicated by the biochemical analysis in Figure 4A. Taken together, the results demonstrated that Fas, which was localized to the Golgi in the undifferentiated state, was shifted to lipid rafts on the cell surface by binding to the HS4C3- binding epitope during differentiation. PLOS ONE | www.plosone.org 11 August 2012 | Volume 7 | Issue 8 | e43440