Sc5279

Whole cell lysates were extracted in RIPA buffer (Sigma) with 1x cOmplete Mini EDTA-free Protease Inhibitor Cocktail (Roche). Proteins were separated by electrophoresis using 12% SDS-polyacrylamide gels and transferred to 0.45 μM PVDF membranes (Amersham Hybond). Membranes were blocked for > 3h in TBS-T 5% milk and hybridized to primary antibody overnight at 4°C. Membranes were washed 3x for 10min in TBS-Tween 5% milk at room temperature then incubated for 1h at room temperature with secondary antibodies HRP-conjugated goat-anti-mouse, goat-anti-rabbit (1:5,000, Bio-Rad) or donkey-anti-goat (1:2,500, Jackson ImmunoResearch) immunoglobulins. Detection was performed using Clarity Western ECL reagent (Bio-Rad). Primary antibodies: TFCP2L1 (1:500, R&D Systems AF5726); KLF17 (1:200, Atlas Antibodies HPA024629); POU5F1 (1:500, Santa Cruz sc5279); β-ACTIN (1:1000, Sigma A5441).

For histology analyses, tissues were fixed in 4% paraformaldehyde (PFA), followed by dehydration, embedded in paraffin and processed into 5 μm sections for staining with haematoxylin (Sigma‐Aldrich, MHS16) and eosin (Sigma‐Aldrich, HT110216). Images were obtained with a Leica microscope (Leica, DM4000B). IHF was performed as previously described by Zhang et al.
²⁷ (link) Briefly, embryonic gonads were fixed with 4% PFA in PBS at 4°C for 2 h and then embedded in paraffin. The 5 μm‐thick gonad sections were incubated with primary antibodies followed by washing three times with PBS containing 0.1% Triton X‐100. Nuclei were stained with DAPI after blotting with fluorochrome‐conjugated second antibodies. Images were obtained with a confocal microscope (Andor‐Oxford Instruments, DF505). Primary antibodies used in this study: OCT4 (used as 1:100 dilution, Santa Cruz, sc‐5279), STELLA (Sigma, 1:300, MAB4388); NRF1 (1:400, Abcam, ab175932), DDX4 (1:400, Abcam, ab27591), Ki67 (1:400, Abcam, ab15580) and cleaved Caspase‐3 (1:400, Cell Signalling Technology, #9661).

All human pluripotent stem cell studies were carried out in accordance with approval from the National University of Singapore’s Institutional Review Board (IRB 12–451). Pluripotent hESCs H1 (karyotype: 46, XY; WiCell Research Institute, WA01, RRID:CVCL_9771) were maintained on 10 μg/ml LN521 (Biolamina AB, LN521)-coated culture plates with daily change of Nutristem® (Biological Industries, 05-100-1 A) medium. Routine monitoring of pluripotent markers POU5F1 (Santa Cruz, sc-5279, RRID:AB_628051) and Tra1–60 (Millipore, MAB4360, RRID:AB_2119183) by flow cytometry and genomic stability by karyotyping were performed at the Singapore General Hospital cytogenetics laboratory. Cells were split at 200,000 cells per well and were passaged at 80% confluence by gentle dissociation with TrypLE (ThermoFisher, 12563011) at 37 °C for 8 mins to dissociate the single cells. The cell suspension was then collected and centrifuged at 800 rpm for 4 mins. Supernatants were discarded and the cell pellets were resuspended in 1 mL of pre-warm Nutristem® medium. Bright-field images were taken with a Leica microscope.