Figure 5
From: Dysregulation of nuclear receptor COUP-TFII impairs skeletal muscle development
COUP-TFII represses FAK activity and directly regulates expression of moyblast fusion-associated genes in C2C12 cells. Quantitative RT-PCR examination of Nephronectin, Integrin β1D and Caveolin 3 expression in (A) Ectopic COUP-TFII overexpressing cells post-serum withdrawal at the indicated time points; and (C) COUP-TFII silencing C2C12 cells one day after serum withdrawal. Total RNA was extracted using TRIzol® reagent and mRNA expression levels were quantified by SyBr Green based RT-qPCR analysis. Western blot examination of indicated protein expression patterns in (B) ectopic COUP-TFII overexpressing and (D) COUP-TFII silencing C2C12 cells during myogenesis. The cells were harvested with 0.1% SDS-RIPA lysis buffer at the indicated time points under serum withdrawal culture condition. Equal amounts of cell lysates were subjected to immunoblotting with the indicated antibodies. Protein expression levels were quantified by densitometry, normalized by GAPDH levels, and shown in the bottom panel as relative values. (E) ChIP-qPCR analysis of the recruitment of COUP-TFII to the Npnt, Itgb1 and Cav3 enhancer region. Bar graph showed the enrichment of DNA fragments pulled down by COUP-TFII antibody. NB: non-binding region. Data are shown as mean ± SEM. Two-tailed Student’s t test; *p < 0.05, **p < 0.01, compared to IgG control.
