Cm163a

Whole mount immunostaining of cultured ovaries was performed as described in (91 ). Briefly, explanted ovaries attached to the polycarbonate membrane were fixed in 2% PFA at 4°C overnight. Fixed ovaries were washed with 70% ethanol overnight and placed in PBS for at least 4 hours before immunostaining. Ovaries were permeabilized in solution containing 0.2% polyvinyl alcohol (PVA), 0.1% NaBH₄, and 1.5% Triton X-100 in PBS for 4 hours, and incubated in blocking solution [10% goat serum, 3% bovine serum albumin, 0.15% glycine, pH 7.4, 0.1% Triton X-100, 0.2% sodium azide, penicillin (100 U/ml), and streptomycin (100 ng/ml), in PBS] overnight. Primary antibody incubation was performed at room temperature with gentle rocking for 2 to 4 days. Primary antibodies used were mouse anti-p63 (4A4, Biocare Medical, CM163A) and rabbit anti-DDX4 (Abcam, ab13840). Ovaries were washed with wash solution (0.2% PVA and 0.15% Triton X-100, in PBS) for 1 to 2 days with buffer changes (2× daily). Ovaries were next incubated with Alexa Fluor secondary antibodies for 2 to 3 days followed by washing for 1 to 2 days with buffer changes.

Cells were lysed in CST lysis buffer (Cell Signaling Technology) supplemented with MG132 (40 μM; Calbiochem) and cOmplete Protease Inhibitor Cocktail (Roche). Lysates were separated by SDS–PAGE and immunoblotting performed on polyvinylidene difluoride transfer membranes (Fisher Scientific) with primary antibodies diluted in 5% BSA overnight at 4 °C. The following antibodies were used for immunoblotting: GLUT1 (1:1,000; Alpha Diagnostic; GT11-A), p63 (1:1,000; Biocare Medical; CM163A), CK5 (1:1,000; Abcam; ab52635), HIF-1α (1:1,000; BD Transduction Laboratories; 610959), AKT and Ser473-p-AKT (1:1,000; Cell Signaling; 9,272 and 9,271, respectively), β-actin (1:5,000; Sigma; A5441). Horseradish peroxidase-conjugated secondary antibodies (Santa Cruz) were diluted 1:5,000 in 5% skim milk, followed by detection with SuperSignal West Femto or Pico substrate kits (ThermoFisher). Uncropped images of immunoblots are provided as Supplementary Fig. 22.