AMPK regulates cAMP-induced steroidogenic gene expression.
A, MA-10 cells were treated with cAMP (100 μm) for the indicated time period; cell lysates were extracted, and a cellular ATP assay was performed. Results are representative of three independently performed experiments. B, MA-10 cells were treated with cAMP (100 μm) for the indicated time period; cell lysates were extracted, and Western blot analyses were performed using the indicated antibodies. Results are representative of three independently performed experiments. C, MA-10 cells were transfected with shPEPCK (200 ng) or pSuper vector (200 ng) prior to 8-Br-cAMP (100 μm) treatment. Total RNA was isolated for semiquantitative RT-PCR analysis. Data represent mean ± S.D. of three individual experiments. D, MA-10 cells were incubated with cAMP (100 μm) and then added 3-MPA (10 μm) or shPEPCK (200 ng); cell lysates were extracted, and a cellular ATP assay was performed. Results are representative of three independently performed experiments. E, MA-10 cells were transfected with shPEPCK (200 ng) or pretreated with 3-MPA (10 μm) prior to 8-Br-cAMP (100 μm) treatment. Cells lysates were extracted, and Western blot analyses were performed using p-AMPK and AMPK antibodies. Results are representative of three independently performed experiments. F, MA-10 cells were pretreated with AICAR (200 or 300 μm) prior to cAMP (100 μm) treatment. Cells lysates were extracted, and Western blot analyses were performed using p-AMPK and AMPK. Results are representative of three independently performed experiments. G, MA-10 cells were infected with the CA-AMPK adenovirus at an m.o.i. (MOI) of 10, 30, or 60 for 24 h followed by cAMP (100 μm) treatment for 12 h. Total RNA was isolated for semiquantitative RT-PCR analysis. Data represent mean ± S.D. of three individual experiments. H, MA-10 cells were treated with AICAR (100, 200, or 300 μm) for 12 h followed by a preceding 12 h cAMP (100 μm) treatment. Total RNA was isolated for semiquantitative RT-PCR analysis. Data represent mean ± S.D. of three individual experiments.