Skip to main content
. 2013 Jan 22;27(3):480–490. doi: 10.1210/me.2012-1347

Figure 4.

Figure 4.

SIRT1 occupies aromatase promoters PII/PI.3 and PI.4 and inhibition causes a decrease in aromatase transcripts and ERRα protein. (A) MDA-MB-231 cells were treated with vehicle (DMSO) or increasing concentrations of cambinol (25μM, 50μM, and 100μM) for 24 hours, and Western blottings for ERRα and β-actin were performed on equal protein. (B) MDA-MB-231 cells were stably infected with either a control or virus targeting 2 distinct regions of ERRα. Western blottings for ERRα (top panel) and semiquantitative RT-PCR were used to monitor changes in aromatase (middle panel). β-Actin is shown as a loading control (bottom panel). (C) (left panel) Subconfluent MDA-MB-231 cells were treated with DMSO or increasing concentrations of cambinol (50μM and 100μM) for 30 minutes, and acetyl-lysine (#9681) was immunoprecipitated followed by Western blottings for ERRα. The antibody light chain (LC) was probed, and a Western blotting for ERRα in whole cell extracts (WCE) was performed. (C) (right panel) Confluent MDA-MB-231 were treated with DMSO or 50μM cambinol for 30 minutes, and acetyl-lysine was immunoprecipitated followed by Western blottings for ERRα. (D) ChIP of MDA-MB-231 cells for SIRT1, ERRα, and rabbit IgG were performed. Primers against the promoter II/I.3 and promoter I.4 were used to determine the presence of both SIRT1 and ERRα at these promoters. E-cadherin primers were also included as a control, because SIRT1 is known to bind to its promoter. The input lane represents sonicated material that was not exposed to antibody immunoprecipitation. (E) ChIP for rabbit IgG and ERRα in MDA-MB-231 cells treated with either vehicle (−) or 100μM cambinol (+) for 3 hours. Primers against the promoter II/I.3 were used to determine the presence of ERRα at this promoter region. The input lane represents sonicated material that was not exposed to antibody immunoprecipitation. Data shown are representative of at least 3 independent experiments. RNAi, RNA interference; Ctrl, control; Veh, vehicle; Cam, cambinol; IP, immunoprecipitation; AC, acetyl.