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. 2009 Oct 1;20(19):4235–4245. doi: 10.1091/mbc.E08-12-1247

Figure 5.

Figure 5.

Suppression of Ad4BP/SF-1 mediated transcription by ARIP4. (A) The effect of Ad4BP/SF-1 or ARIP4 depletion on StAR promoter activity. MA-10 cells were transfected with siRNAs as indicated. pREP-hStAR-Luc was used as a reporter gene (200 ng), and 1 mM 8Br-cAMP was added 24 h later. The relative fold changes in the luciferase activities are plotted, with the activity of Ad4BP/SF-1 stimulated by 8Br-cAMP in the presence of control siRNA set at 100. Values are indicated as the means ± SD of at least three experiments. (B) The effect of Ad4BP/SF-1 or ARIP4 depletion on target gene expression. After MA-10 cells were treated with siRNA (as described above), the mRNA for Cyp11a1, StAR, and Inhibin α was examined with RT-PCR. As a control, Control siRNA was used. The relative fold changes are plotted, with the amounts of mRNA in the presence of the control siRNA set at 100. Values are indicated as the means ± SD of at least three experiments. (C) The effect of ARIP4 on Ad4BP/SF-1–mediated transcription of the Cyp11A1 (pREP-Cyp11A1-Luc) and StAR (pREP-hStAR-Luc) genes. HEK293 cells were transiently transfected with the indicated amounts (ng) of pCMX-Ad4BP/SF-1 (Ad4BP), pCMX-Ad4BP/SF-1 K119R/K194R mutant (KR), and pcDNA-ARIP4 (ARIP4). Luciferase reporter genes were cotransfected. After 24 h, 20 μM forskolin was added. After another 24 h, the total cell lysates were recovered. The relative fold changes in the luciferase activities are plotted, with the activity of Ad4BP/SF-1 or KR mutant in the absence of ARIP4 set at 100, respectively. Values are indicated as the means ± SD of at least three experiments. (D) The effect of hARIP4 ATPase mutation (K311A). pREP-hStAR-Luc was cotransfected into Y-1 cells with wild-type or ATPase mutant hARIP4. The cells were stimulated by 10 nM ACTH, and the luciferase activity was determined 24 h later. The relative fold changes in the luciferase activities are plotted, with the activity of Ad4BP/SF-1 in the absence of ARIP4 set at 100. Values are indicated as the means ± SD of at least three experiments. (E) Differential effect of ARIP4 on transcription mediated by wild-type and KR mutant form of Ad4BP/SF-1. HEK293 cells and pREP-hStAR-Luc were used for the study. pCMX-Ad4BP/SF-1 (solid and open circles) or pCMX-Ad4BP/SF-1 KR mutant (solid and open square) was transfected to HEK293 cells with siRNA for ARIP4 (open cycle and square) or control siRNA (solid circle and square). After 18 h incubation, the cells were treated with 2.5 μM α-amanitin for 2 h, and washed twice with PBS. The cells were incubated with serum-free medium containing 20 μM forskolin. Total RNAs were prepared from the cells 0, 30, 60, or 120 min after the forskolin treatment. Luciferase mRNAs were quantified with real time PCR. The relative mRNA levels are plotted when the amounts of the mRNAs at time 0 are fixed as basal level. Values are indicated as the means ± SD of at least three experiments. (F) Transient recruitment of ARIP4 to Ad4BP/SF-1 target gene promoter. HEK293 cells were transfected with pREP-hStAR-Luc and pCMX-Ad4BP/SF-1 (solid bar) or pCMX-Ad4BP/SF-1 KR (K119R/K194R) mutant (open bar). After α-amanitin treatment for 2 h and washing twice with PBS, the cells were incubated with serum-free medium containing 20 μM forskolin. Fixed chromatins were prepared from the cells at the time points indicated, and subjected to immunoprecipitation with anti-Ad4BP/SF-1 antibody. The recovered chromatin fraction was further subjected to immunoprecipitation with anti-ARIP4 antibody. The StAR gene promoter recovered in the two precipitated fractions was quantified by PCR. The amounts of StAR gene promoter recovered by ARIP4 antibody relative to those by Ad4BP/SF-1 antibody (ARIP/Ad4BP/SF-1 or ARIP4/KR) were calculated. The relative fold changes of the relative values above are plotted, with the amount in the presence of wild-type Ad4BP/SF-1 at 0 min incubation set at 1.0. Values are indicated as the means ± SD of at least three experiments. (G) Transient recruitment of ARIP4 to the endogenous Mc2R promoter. Y-1 cells were cultured in a serum-free medium for 48 h and treated with 2.5 μM α-amanitin for 2 h. After ACTH treatment, soluble chromatin was prepared from the cells at the time points indicated, and then subjected to ChIP assay with anti-Ad4BP/SF-1, anti-ARIP4 antibody, or control antibody. The recovered chromatin was amplified using primers for the proximal or distal Mc2R promoter.