FIG 7.

Knockdown of NGFIB and NURR1 attenuates AngII-stimulated HSD3B1 induction. (A) Gene-selective knockdown by electroporation of siRNAs against NGFIB and NURR1. H295R cells were transfected by electroporation using the indicated siRNA mixtures, each directed against NGFIB (siNGFIB) or NURR1 (siNURR1), and treated with 100 nM AngII or vehicle for 4 h. Total RNA was isolated, and expression of NGFIB and NURR1 was determined by qRT-PCR. All values (means ± SEMs, n = 3) were normalized to the levels of RPLP0. As a control, the electroporation was also performed with a negative-control siRNA (NC). (B) Reduction of protein expression of NGFIB and NURR1 by siRNA-mediated knockdown. H295R cells transfected with the indicated siRNAs were stimulated by 4 h AngII treatment (10 nM or 100 nM) and subjected to Western blotting with antibodies against NGFIB and NURR1. α-Tubulin was used as a loading control. (C) Attenuated AngII response of HSD3B1 and CYP11B2 by knockdown of NGFIB and NURR1. H295R cells were transfected with the indicated siRNAs and then stimulated with either 100 nM or 10 nM AngII or vehicle for 4 h. The levels of HSD3B1 and CYP11B2 mRNA were determined by qRT-PCR (n = 3 for each), and the means of vehicle treatment were set equal to 1 after normalization to the level of RPLP0. Error bars indicate SEMs. *, P < 0.05 (versus 10 nM AngII induction of negative-control siRNA-transfected cells); #, P < 0.05 (versus 100 nM AngII induction of negative-control siRNA-transfected cells). (D) Unimpaired AngII response of HSD3B1 by electroporation of siRNAs targeting NOR1 (siNOR1). H295R cells transfected with the indicated siRNAs (s15541, s15543, or the negative-control siRNA) were treated with 100 nM AngII or vehicle for 4 h. Total RNA was isolated, and expression of NGFIB, NURR1, NOR1, and HSD3B1 was determined by qRT-PCR. All values (means ± SEMs, n = 3) were normalized to the level of RPLP0.