Fig. 5.

SHOX2 and STAT3 are assembled in the same complex on the WASF3 promoter. (a) CpG methylation level of the WASF3 promoter in SHOX2 overexpressing (SHOX2) and control (EV) T47D cells. Individual clones (n = 12) for each cell line were sequenced, and the methylation level of CpGs was analyzed based on the sequencing data (open circle, unmethylated CpG; closed circle, methylated CpG). (b) The accumulation of STAT3 on the WASF3 promoter was determined by ChIP-QPCR assays in SHOX2 overexpressing and control T47D cells. (c) The binding of SHOX2 to STAT3 was determined by IP assays in SHOX2 overexpressing T47D cells. (d) The effect of 50 µM S3I-201 on SHOX2-STAT3 interaction in overexpressing T47D cells. In (c) and (d), cell lysates were IP using an anti-FLAG antibody and immunoblotted with anti-STAT3 and anti-SHOX2 antibodies. IgG was served as a negative control. (e) The binding of the SHOX2/STAT3 complex on the WASF3 promoter was determined by ChIP and re-ChIP assays in SHOX2 overexpressing and its control T47D cells. Chromatin fragments immunoprecipitated with anti-SHOX2 antibody in the first ChIP were used as an input and were subjected to re-ChIP analysis using control IgG or anti-STAT3 antibody. ^*p < 0.05, ^***p < 0.001