Oct3 4

Cells were lysed in RIPA with 1 mM PMSF for 30 min on ice. The mixture was centrifuged at 14,000 g for 5 min and the precipitate was discarded. BCA protein assay (Beyotime, China) was performed to measure the protein concentration. Samples containing equal amount of protein were separated by SDS-PAGE and electroblotted onto polyvinylidene fluoride (PVDF) membrane using standard procedure. Non-specific sites were blocked with 5% nonfat milk for 1 hour at room temperature. PVDF membrane was incubated at 4°C overnight with corresponding primary antibodies, Nanog(Cell Signaling Technology, Beverly, MA, USA), Oct3/4(Cell Signaling Technology, Beverly, MA, USA), STAT3(Cell Signaling Technology, Beverly, MA, USA), ABCG2(Cell Signaling Technology, Beverly, MA, USA), BCL-2(Cell Signaling Technology, Beverly, MA, USA, Beverly, MA, USA),and DNA-PKcs(Abcam, Cambridge, UK). After washed in TBS with 0.1% Tween 20, blots were incubated with anti-rabbit or anti-mouse secondary antibodies conjugated with horseradish peroxidase. Immunoreactive bands were detected by enhanced chemiluminescence(ECL)(GE Healthcare, UK).

Cells grown on 10% HSt-PMEDSAH-g dishes were fixed in 4% paraformaldehyde for 10 min at room temperature followed by permeabilized with 0.1% Triton X-100 for 10 min. Primary antibodies raised against OCT3/4 (Cell signaling), SOX2 (MilliporeSigma, Burlin-tong, MA, USA), NANOG (Abcam, Cambridge, UK), TRA-1-60 (Abcam), and TRA-1-81 (MilliporeSigma) were diluted in 1% normal donkey serum and incubated overnight at 4 °C with gentle shaking and detected with respective secondary antibodies. Micrographs were captured using an EVOS®FL Cell Imaging System (Thermo Fisher Scientific).

Quantitative western blot analysis using a radioactive detection method was performed as previously described [15 (link)]. Primary antibodies used included proliferating cell nuclear antigen (PCNA), RXRα, RARα, ACTIN (Santa Cruz Biotechnology, Santa Cruz, CA), cellular retinoic acid-binding protein II (CRABPII), cytochrome P450 26A1 (CYP26A1), PPARβ/δ (Abcam, Cambridge, MA), octamer-binding transcription factor 3/4 (OCT3/4; Cell Signaling Technology, Danvers, MA), fatty acid binding protein 5 (FABP5; BioVedor Inc., Asheville, NC) and lactate dehydrogenase (LDH; Rockland, Gilbertsville, PA), The expression level of each protein was normalized to LDH or ACTIN.