HS4C3-Binding Epitope Contributes to Fas Signaling withdrawal up to day 6. The values shown are means 6 SD. (D) FACS analysis, using the HS4C3 antibody, of mESCs at 2 days after transfection with the 3OST-5 expression construct. In the left panel, a histogram shows a representative result of the FACS analysis (black line, control cells; red line, cells overexpressing 3OST-5). The gray line shows the result obtained from cells not treated with primary antibody. The control cells were transfected with empty pCAGI vector. In the right panel, the values shown are the mean fluorescence intensity 6 SD after normalization against control cells (arbitrary value = 1). (E) Self-renewal assay with cells overexpressing 3OST-5. Left panels show photographs of representative colonies. A high magnification image is shown at the bottom of each photograph to the right. Scale bars, 200 mm. 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 or without LIF. (F) and (G) Real time PCR analysis of markers of the undifferentiated (F) and differentiated state (G) in mESCs at 4 days after transfection with the 3OST-5 expression construct. The values shown are means 6 SD after normalization against control cells (arbitrary value = 1). DIC, Differential interference contrast. *, P,0.01; **, P,0.05. At least three independent experiments were performed in each case. doi:10.1371/journal.pone.0043440.g001 FACS Analysis Cells harvested 2 days after transfection were incubated with a vesicular stomatitis virus (VSV)-tagged phage-display antibody against specific sulfated HS structure [30], in FACS buffer (0.5% bovine serum albumin {BSA} and 0.1% sodium azide in PBS). After washing, the cell suspension was incubated with mouse anti-VSV glycoprotein antibody (Sigma) in FACS buffer, washed, and stained with a Cy5-conjugated anti-mouse IgG antibody (Jackson ImmunoResearch) or FITC-conjugated anti- mouse IgG antibody (Sigma) in FACS buffer. Cell analysis was performed using a FACSAria Cell Sorter (Becton Dickinson). We used the VSV-tagged HS4C3 antibody to analyze 3-O- sulfated HS structures [31]. We also used the anti-HS antibody 10E4 (Seikagaku Corp.) and FITC-conjugated anti-mouse IgM as the second antibody (Sigma) for antibody 10E4. We analyzed the expression of Fas using the FITC-conjugated anti-Fas antibody Jo2 (Becton Dickinson). The cells were permeabilized in 70% ethanol for 30 min on ice before Fas staining to enable analysis of total Fas expression [32]. The effect of the inhibition of protein secretion on Fas expression was examined using cells that had been treated with 5 mg/ml brefeldin A (Sigma) for 2 h before harvest. Measurement of Viability Two days after transfection, cells were harvested and replated in triplicate at 0.86104 cells per well in 96-well 0.2% gelatin-coated plates in ESC medium with LIF. Cell Counting Kit-8 solution (Dojindo) was added after 0, 24 or 48 h and the cells were incubated for a further 2 h. The resulting soluble formazan product, which reflects the number of living cells, was measured at 450 nm. Viability was indicated by the ratio of the absorbance of the transfected cells to that of control cells. Measurement of Apoptotic Cells Two days after transfection, cells were harvested and suspended in annexin V-binding buffer (BioVision). Annexin V-FITC (BioVision) was added to the cell suspensions, which were incubated at room temperature for 5 min in the dark. The suspensions were analyzed using a FACSAria Cell Sorter. Analysis of Proteins by Immunoblotting Two days after transfection, protein samples for immunoblot- ting were prepared as follows. To analyze phosphorylated protein in transfected cells after extrinsic stimulation, mESC culture medium was replaced with serum-free ESC medium without LIF for 4 h and the cells were stimulated for 5, 10 or 20 min with FBS or 5 min Fgf4. Cells were then lysed with lysis buffer (50 mM Tris- HCl pH 7.4, 150 mM NaCl, 1% Triton X-100, 1 mM Na3VO4, 10 mM NaF, protease inhibitors). Lipid rafts were isolated as described previously [33,34,35]. Harvested cells were suspended in 0.5 ml of homogenization buffer (50 mM Tris-HCl pH 6.5, 150 mM NaCl, 5 mM EDTA, 1 mM Na3VO4, 10 mM NaF, protease inhibitors) and homogenized by passing through a 20-G needle 50 times on ice. After adding 0.5 ml of lysis buffer (50 mM Tris-HCl pH 6.5, 150 mM NaCl, 5 mM EDTA, 1.2% Triton X- 100, 1 mM Na3VO4, 10 mM NaF, protease inhibitors) to the suspension, it was homogenized again by passing through a 20-G needle 10 times and incubated for 30 min on ice. The extract (approx. 1 mg of total protein) was mixed with 1.0 ml of 85% sucrose (Wako) to produce a 42.5% sucrose solution, transferred to a centrifuge tube (Beckman Coulter), and overlaid with 5 ml of 30% sucrose solution and 3 ml of 5% sucrose solution containing 50 mM Tris-HCl pH 6.5, 150 mM NaCl, and 5 mM EDTA. The discontinuous sucrose gradients were centrifuged at 4uC for 16 h in an SW41 Ti rotor (Beckman Coulter) at 30,000 rpm. The gradient was divided into nine fractions from the bottom to the top. The proteins in each fraction were precipitated with 10% trichloroacetic acid and washed with 5% trichloroacetic acid, followed by cold acetone. The precipitate was dried and dissolved in lysis buffer. Aliquots of 10 mg of total protein from the cell samples or half the total protein from each fraction were separated by 10% or 15% SDS-PAGE and transferred onto PVDF membranes (Millipore). After blocking, the membranes were incubated with antibodies against cleaved (activated) caspase-3, cleaved (activated) caspase-8, uncleaved (unactivated) caspase-8, Bad, Erk1/2, phosphorylated Erk1/2 (Thr202 and Tyr204) (Cell Signaling Technology); b-actin (Sigma); Akt, phosphorylated Akt (Ser472 and Ser473), Flotillin-1 (Becton Dickinson); Fas (M-20) (Santa Cruz); transferrin receptor (Zymed); or Nanog (Repro- CELL). The membranes were then incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling Technology), washed, and developed with ECL Plus reagents (GE Healthcare). Binding Assay The putative extracellular domain of mouse Fas or fragments thereof (amino acids 19–168, 19–38, and 39–168, lacking the signal peptide) and the domains that included point mutations (K32A, R34A, R35A, R36A, and H38A) were expressed in Escherichia coli BL21 cells as fusion proteins with gluthathione sepharose transferase (GST), and purified with gluthathione sepharose 4B resin (GE Healthcare) according to the manufac- turer’s instructions. The K32A, R34A, R35A, R36A, and H38A point mutants were generated from the template pGEX-6P-1-Fas (amino acids 19–168) using the KOD Plus Mutagenesis Kit (Toyobo) according to the manufacturer’s instructions. Fas binding activity was examined by an overlay assay as described previously [36]. Fas overlay assays were performed on a PVDF membrane using the purified GST-Fas fusion protein. Samples (2.5 mg of total protein) were separated by 8% SDS-PAGE and then transferred to PVDF membranes. The membranes were blocked in blocking buffer (5% non-fat dry milk in PBS pH 6.5), and then incubated with 10 nM GST-Fas, GST-mutated Fas, or GST-Fas pre-mixed with HS4C3 (1:1000) for 2 h at 4uC in PBS pH 6.5 with 3% BSA. The membranes were washed in buffer (PBS pH 6.5, 0.1% Tween PLOS ONE | www.plosone.org 4 August 2012 | Volume 7 | Issue 8 | e43440