Figure 3.

A, HESCs were transfected with siRNA targeting ESR1 or scrambled siRNA (control [siCTRL]). Forty-eight hours after transfection, HESCs were treated with a differentiation cocktail to initiate in vitro differentiation. Cells were harvested at 24 and 72 hours after the differentiation cocktail treatment. Total RNA was isolated and cDNAs were prepared. cDNAs were subjected to real-time PCR using gene-specific primers for 36B4, PGR, FOXO1, and WNT4. PCR data are normalized to 36B4 mRNA levels. Relative fold changes were normalized with respect to control nontargeting siRNA treatment. Mean ± SEM of three independent experiments are shown. The Student's t test was used for statistical analysis. **, P ≤ .01; *, P ≤ .05; ns, nonsignificant. B, HESCs were hormone starved in 2% charcoal-stripped serum containing medium for 48 hours. Then cells were either untreated or treated with E alone, E+P, or E+P+cAMP for 24 hours. Total RNA was isolated and cDNAs were prepared. cDNAs were subjected to real-time PCR using gene-specific primers for 36B4, FOXO1, and WNT4. PCR data are normalized to 36B4 mRNA levels. The figure shows mean ± SEM of three independent experiments. A one-way ANOVA followed by a Tukey honest significant difference test was used for statistical analysis. **, P ≤ .01; *, P ≤ .05; ns, nonsignificant.