Protein Expression Analysis in JEG-3 and BeWo Cells

The levels of protein expression were measured using previously reported methods (12 (link)). Total proteins from the treated JEG-3 and BeWo cells were isolated using Radio-Immunoprecipitation assay (RIPA) buffer with 1% Phenylmethanesulfonyl fluoride (PMSF) (Abcam, Cambridge, UK). The protein concentrations were quantified using the Bicinchoninic acid (BCA) protein assay kit (Keygen, Beijing, China). The protein levels of the target genes were determined by Western blot analysis. The working concentration of primary anti-β-actin (ACTB) (Abcam, Cambridge, UK) was 1:2,000. The working concentrations of primary anti-cleaved Poly ADP-ribose polymerase (PARP) (Abcam, Cambridge, UK), anti-p21 (Abcam, Cambridge, UK), anti-MDM2 (Abcam, Cambridge, UK), anti-BCL2 (Abcam, Cambridge, UK), and anti-BAX (Abcam, Cambridge, UK) were 1:1,000. The working concentrations of primary anti-HIF1α (Abcam, Cambridge, UK) and anti-p53 antibody (Abcam, Cambridge, UK) were 5 μg/mL. The relative amount of each target protein was normalized to ACTB.