Figure 8.

Silencing of DAX-1 and its relevance to StAR expression and progesterone synthesis. MA-10 cells were transfected with either a negative control siRNA at 100 nm (Con siRNA) or a mixture of two DAX-1-specific siRNAs at 50 nm (100 nm total, DAX-1 siRNA), as described in Materials and Methods. After 36 h of transfection, cells were treated without (Con) or with (Bu)₂cAMP (0.5 mm) and PMA (10 nm) for an additional 6 h and subjected to Western (DAX-1, StAR, and actin) and RT-PCR (StAR, DAX-1, and L19) analyses. Representative immunoblots and autoradiograms illustrate the expression of DAX-1, StAR, and actin (A) and StAR, DAX-1, and L19 (B) in different treatment groups, respectively. Actin and L19 were used as controls in Western and RT-PCR analyses, respectively. Similar results were obtained from three different experiments. C, Accumulation of progesterone in the media was determined and expressed as nanograms per milligram protein, and the data shown represent the mean ± se of three independent experiments. Letters above the bars indicate that these groups differ significantly from each other, at least at P < 0.05.