HS4C3-Binding Epitope Contributes to Fas Signaling histograms show the ratio of the HS4C3 and annexin V double positive cells to control cells (left graph) or mESCs cultured in the presence of LIF (+LIF) (right graph) (value = 1). Values are the means 6 SD. (D) and (E) Western blot analysis, using antibodies against cleaved caspase-3 or Nanog, of mESCs at 2 days after transfection with the 3OST-5 expression construct (D, left two lanes and E) or of mESCs cultured for 8 days in the absence of LIF (2LIF) (D, right two lanes). The histograms show mean densitometric readings 6 SD after normalization against control cells (D, left two lanes and E), or mESCs cultured in the presence of LIF (+LIF) (D, right two lanes) (arbitrary value = 1). (F) Real time PCR analysis of Nanog in mESCs at 2 days after transfection with the 3OST-5 expression construct. The values shown are means 6 SD after normalization against control cells (arbitrary value = 1). *, P,0.01; **, P,0.05. Three independent experiments were performed in each case. doi:10.1371/journal.pone.0043440.g002 20) and incubated with HRP-conjugated anti-GST antibody (Santa Cruz) for 1 h at room temperature. Blots were developed with ECL Plus reagents. RT-PCR and Real Time PCR Total RNA was isolated from cells using TRIzolH Reagent (Invitrogen) and reverse transcribed using an oligo-dT primer (Invitrogen) and a Superscript II First Strand Synthesis Kit (Invitrogen). Real time PCR was performed using an ABI PRISMH 7700 Sequence Detection System (Applied Biosystems). The relative amounts of each mRNA were normalized against the amount of b-actin or GAPDH mRNA in the same sample. Primer sets and probes for real time PCR are listed in Tables S1 and S2, respectively. Self-Renewal Assay Two days after transfection of pCAGI that contained 3OST-5 or Fas, mESCs were replated at 16104 cells per gelatin-coated 60-mm tissue culture dish in ESC medium with or without LIF. In the case of treatment with the inhibitors IETD and DEVD, the mESCs were treated throughout the period from transfection to alkaline phosphatase (AP) staining. To detect undifferentiated cells, cells were fixed and stained with 5-bromo-4-chloro-3- indolyl phosphate-nitroblue tetrazolium (Nacalai Tesque) 5 days after replating. AP-positive colonies were counted by microscopy. Immunostaining of mESCs after Induction of Differentiation into Primitive Endoderm mESCs were fixed with 4% paraformaldehyde and washed with PBS. Subsequently, the cells were blocked with buffer1 (5% BSA, 1% normal goat serum in PBS). After washing with buffer1, the cells were incubated with HS4C3 antibody in buffer1. Then, the cells were washed again and incubated with mouse anti-VSV glycoprotein antibody (Sigma). Finally, the cells were washed and costained with Cy5-conjugated anti-mouse IgG (Jackson Immu- noResearch) and the FITC-conjugated anti-Fas antibody Jo2 in buffer1. To permeabilize the cells, 0.05% Triton X-100 was added to buffer1 throughout the process. Immunofluorescence images were taken using an LSM 5 Pascal confocal laser scanning microscope (Carl Zeiss) with a 63 6/1.3 objective at room temperature. Results Increase in HS4C3-Binding Epitope Induces Differentiation First, we examined changes in HS chain sulfation on the surface of mESCs after induction of differentiation into primitive endoderm by LIF withdrawal. The differentiated cells displayed a reduction in the expression of Nanog and an increase in the expression of Gata6, which is a marker of the primitive endoderm (data not shown). Fluorescence-activated cell sorting (FACS) using the anti-3-O-sulfated HS antibody HS4C3 showed that the HS4C3-binding epitope increased steadily in differentiated mESCs (Figure 1A). Indeed, immunostaining of non-permeabi- lized cells showed that the HS4C3-binding epitope increased on the surface of differentiated mESCs in the absence of LIF for 7 days (Figure 1B). The expression of 3OST-5, which has been reported to synthesize the HS4C3-binding epitope, increased when mESCs were induced to form primitive endoderm by the withdrawal of LIF (Figure 1C). Next, we performed FACS analysis of cells transfected with an expression construct for 3OST-5 using the antibody HS4C3. The HS4C3-binding epitope was increased in cells overexpressing 3OST-5, but the amount of HS did not differ between the cells overexpressing 3OST-5 and the control cells (Figure 1D and Figure S1). To determine whether overexpression of the HS4C3-binding epitope induced differentiation of mESCs, we performed a self- renewal assay with cells overexpressing 3OST-5. The number of AP-positive colonies that were derived from cells overexpressing 3OST-5 decreased to approximately 30% of the number obtained with control cells even in the presence of LIF and serum in clonal density culture, which demonstrated a reduction of self- renewal in cells overexpressing 3OST-5 (Figure 1E). Then, we examined the expression of Oct3/4 and Nanog in cells overex- pressing 3OST-5 that were cultured with LIF; Oct3/4 and Nanog are markers of the undifferentiated state. Expression of both Oct3/4 and Nanog was significantly lower in cells overexpressing 3OST-5 than in control cells that were cultured with LIF (Figure 1F). Thus, overexpression of the HS4C3-binding epitope induced differentiation of mESCs. To characterize the differen- tiating mESCs, we evaluated the expression of several germ layer markers. In cells overexpressing 3OST-5, we detected higher expression of markers of the primitive endoderm (Gata6) and primitive ectoderm (Fgf5) than in control cells (Figure 1G). Other lineage markers (neuroectoderm, Isl1; mesoderm, Brachyury; primitive endoderm, Gata4; parietal endoderm, LamininB1; visceral endoderm, Bmp2; trophoblast, Cdx2) were either expressed at a lower level or were unaltered as compared with control cells that were cultured with LIF (Figure 1G). These results demonstrate that the increase in the HS4C3-binding epitope induced differentiation into primitive endoderm and primitive ectoderm during culture in the presence of LIF, which should maintain the undifferentiated state. Activation of Caspase-3 and Degradation of Nanog are Induced in Cells Overexpressing 3OST-5 Next, we examined cell viability because the cell number for cells overexpressing 3OST-5 was low compared with that for control cells. Indeed, cells overexpressing 3OST-5 displayed decreased viability, which might be caused by apoptosis (Figure 2A). Cell differentiation and apoptosis are linked, and mESCs undergo caspase-mediated apoptosis during differentiation induced by LIF withdrawal [37]. As shown in Figure 2B, the proportion of annexin V-positive cells increased significantly in cells overexpressing 3OST-5 as compared with control cells. In addition, the ratio of annexin V and HS4C3 double-positive cells was approximately five times higher in cells overexpressing 3OST- 5 and mESCs induced to differentiate by culture in the absence of LIF for 8 days than in control cells (Figure 2C). These results PLOS ONE | www.plosone.org 6 August 2012 | Volume 7 | Issue 8 | e43440