Figure 5

Involvement of Tbx3 in the transcriptional regulation of Chrna9 in pluripotent stem cells. (A) ChIP assays showing that Tbx3 occupied its binding site on the promoter region of Chrna9. Assays were performed on ES cell extracts using control IgG or anti-TBX3 antibody. Recovered chromatin was subjected to qPCR analysis using primers encompassing proximal Tbx3 binding site (− 0.4 kb) or 5 kb from the transcription start site (− 5 kb). ^⁎P < 0.05 between the indicated groups. (B) Expression of Tbx3 during differentiation of ES cells into mesenchymal cells. Expression of Chrna9 gene was analyzed by qPCR and normalized to 36B4 expression. Data represent the mean ± SEM of four independent experiments. *P < 0.05 vs. day 0. (C) Western blot analyses were conducted in ES cells before (D0) and after differentiation (D5), using antibodies against Tbx3 and Gapdh. (D) Expression of Tbx3 was analyzed in MEF and iPS cells by qPCR and normalized to 36B4 expression. Data represent the mean ± SEM of four independent experiments. Differences between groups are indicated by *P < 0.05. (E) Effects of siRNA-mediated Tbx3 knockdown on the promoter activities of Chrna9 gene. Each reporter construct was co-transfected to ES cells with control siRNA (white boxes) or Tbx3 siRNA (black boxes). To investigate the effects of Tbx3 knockdown, luciferase assays were performed using the cell lysates at 48 h after transfection. Relative luciferase activities are shown. Data are the mean ± SEM values of at least four independent experiments; and *, P < 0.05. (F, G) Effects of siRNA-mediated Tbx3 knockdown on the expression of Tbx3 (F) and Chrna9 (G) in ES cells. Expression of each gene was analyzed by qPCR and normalized to 36B4 expression. Data represent the mean ± SEM of four independent experiments. Differences between groups are indicated by *P < 0.05.