p22PHOX and NOX4 knockdown perturbs cAMP-dependent HESC decidualization. A, Cultured HESCs were untreated or treated with 8-br-cAMP for 6, 8, 10, 16, and 24 h. Whole-cell lysates were extracted and blotted for p22PHOX and NOX4 expression. β-Actin served as a loading control. B, Validation of p22PHOX knockdown at mRNA (left) and protein level (right) in HESCs transfected with NT or p22PHOX siRNAs. C, Quantitative (RTQ-PCR) analysis for both PRL and IGFBP1 levels, normalized to L19 mRNA upon p22PHOX silencing. D, Validation of NOX4 knockdown at mRNA (left) and protein level (right) in HESCs transfected with NT or NOX4 siRNAs. E, RTQ-PCR analysis for both PRL and IGFBP1 levels, normalized to L19 mRNA upon NOX4 silencing. Data in C and E are depicted as fold change relative to transcript levels of samples transfected with NT siRNA and represent means (±sd) of triplicate determinations. F, Primary cultures were transfected with NT siRNA or siRNA targeting p22PHOX and NOX4 and treated 2 d later with 8-br-cAMP for 24 h. The cells were then loaded with DCF-DA for 30 min and DCF fluorescence was measured over a 30-min period. *, P < 0.05; **, P < 0.01; ***, P < 0.001.