Elevated Sox2 alters FoxO1 phosphorylation and its binding to regulatory regions of the <i>Sox2</i> gene.

<p>i-OSKM-ESC were seeded at 1.5×10⁶ per 100 mm dish with or without 4 μg/ml Dox for 24 hours. (A) After the initial 24 hours, the cells were refed with fresh medium with or without 4 μg/ml Dox, and treated with 5 μM AKTiV for an additional 24 hours where indicated. 48 hours after the cells were plated, nuclear and cytoplasmic protein extracts were prepared and equal amounts of nuclear and cytoplasmic protein were loaded into each well of an SDS-PAGE. Western blot analysis was performed by sequentially probing for pFoxO1(S253), total FoxO1, Sox2, HDAC1, and c-Myc. HDAC1 was used as a loading control for two separate western blots performed with the same protein extracts. c-Myc was used as a loading control to confirm proper separation of nuclear and cytoplasmic extracts. The levels of c-Myc in nuclear and cytoplasmic extracts were monitored simultaneously in western blots that were imaged together. (B) Schematic of the location of FBE1, FBE2, and the Sox2 control region within the regulatory regions of the <i>Sox2</i> gene. (C) Chromatin immunoprecipitation (ChIP) analysis of DNA immunoprecipitated from untreated or Dox-treated i-OSKM-ESC using two different anti-FoxO1 antibodies (#1: Cell Signaling Technology, #2: Santa Cruz) and an IgG (GFP) control antibody for normalization. Analysis was performed in triplicate by qPCR and the values for the FoxO1 immunoprecipitated DNA were normalized to the input samples and the control antibody. The fold change of FoxO1 binding was determined by comparing FoxO1 antibody immunoprecipitation of FBE1 and FBE2 to immunoprecipitation of the Sox2 control region from untreated and Dox-treated i-OSKM-ESC. The results are presented as averages of the triplicate results and error is presented as the standard error of the mean. These findings are statistically significant with p-values all <0.05, as determined by Student's t-test.