Fig. 3.

Effect of rapamycin on forskolin-stimulated CREB, StAR, and steroidogenic enzyme expression. Cells were pretreated without or with rapamycin (20 nm) for 1 h followed by forskolin (10 μm) treatment for 24 h. Control groups were treated with vehicle (dimethylsulfoxide). A, The cell lysates were examined for downstream target of mTORC1, eIF4E, CREB, StAR, and steroidogenic enzyme (P450scc, HSD3B1, and P450c17) expression by Western blot analysis. β-Tubulin or GAPDH was used as loading control. The graphs (B–G) represent densitrometric scans of eIF4E, HSD3B1, CREB, StAR, P450scc, and P450c17 protein expression normalized for β-tubulin or GAPDH as seen in A. The blots in A represent one of three separate experiments, and the graphs represent the mean of three experiments. Error bars represent mean + se. **, P < 0.01, ***, P < 0.001 vs. control. a, and b represent significant differences (P < 0.05 and P < 0.01, respectively), compared with FSK. FSK, Forskolin; R, rapamycin.