Anti sox17

The piPSCs were digested into single cell suspension, seeded into low-adhesive 10 cm plate and cultured in embryoid body forming medium consisting of DMEM with 10% FBS, 1% NEAA, 1% GlutaMAX-L and 1% penicillin/streptomycin (Gibico). Low-adhesive culture plates were placed on a shaker (40 r/min) in a CO₂ incubator. After two days, the embryoid bodies were seeded into 1% gelatin-coated plate and cultured in EB forming medium for 10 more days, then fixed for immunocytochemistry. Antibody anti-Neuron specific β III Tubulin antibody (Abcam, ab18207), anti-α-Smooth muscle actin (SMA) (Abcam, ab5694) and anti-Sox17 (Millipore, 09–038) were used for detection of 3 germ-layer-cells. For teratoma formation, the piPSCs (2×10⁷) were subcutaneously injected into the BALB/c nude mice. After two months, the mice were sacrificed by carbon dioxide (CO₂) inhalation. The teratoma were dissected and fixed in 4% PFA. The fixed samples were embedded in paraffin and sections were hematoxylin and eosin staining.

Cells were cultured at a density of 1.5 × 10⁵ cells/well on 8 mm coverslips in 12-well plates. After 48 hours, coverslips were fixed by ice-cold methanol, and incubated with primary E-cadherin (Abcam), Vimentin and β-catenin (Epitomics) antibodies prior to florescent-labeled secondary antibodies. Nuclear DNA was stained with 4′, 6-diamidino-2-phenylindole (DAPI) and coverslips were mounted with FluorSave reagent (CALBIOCHEM). Immunofluorescence images were taken by Olympus inverted fluorescence microscope and were outputted by PV10-ASW 1.7 viewer software. Immunoblotting was performed as follows: Proteins were extracted with lysis buffer and then quantified by the BCA method (KeyGen Biotech). Lysates were diluted in SDS sample buffer (KeyGen Biotech) prior to SDS-PAGE, and then transferred to a polyvinylidene difluoride membrane (Roche Applied Sciences). Membranes were immunoblotted overnight at 4°C with anti-SOX17 (Millipore), anti-CyclinD1 (Abclonal), anti-C-myc, anti-DKK1 (Cell Signaling Technology), anti-E-cadherin (Abcam), anti-SOX2, anti-β-catenin, anti-Vimentin and anti-Slug and anti-N-cadherin antibodies (Epitomics), followed by the appropriate second antibodies. The bands were exposed using Pierce ECL Western Blotting Substrate (Thermo Scientific). Gel densitometry (Bio-Rad) was used to quantify immunoblot signals on exposed film.

Proteins were extracted with RIPA buffer (Sigma-Aldrich) supplemented with a cocktail of protease and phosphatase inhibitors (Sigma-Aldrich). Briefly, cells were trypsinized from culture dishes, centrifuged, and washed once with cold PBS. Cell pellets were then resuspended and incubated in RIPA buffer for 20 min on ice and sonicated with 3 pulses 10 sec each at 10% amplitude (Branson Digital Sonifier, Danbury, CT, USA). After centrifugation, supernatants were denatured in Laemmli loading buffer for 10 minutes at 98°C. Total protein (20 μg) was separated by SDS-PAGE and transferred to PVDF membranes which were subsequently probed with anti-Nanog (Bethyl, Montgomery TX, USA), anti-Oct4 (BD Transduction, San José, CA, USA), anti-Pdx1 (Abcam, Cambridge, UK), anti-Sox17 (Millipore, Billerica, MA, USA), or anti-Gata4 (Santa Cruz Biotechnology, Dallas, TX, USA), and anti-β-actin (Sigma-Aldrich) as loading control.