Figure 3

Modulation of Twist1 expression by Sox2 in RU cells but not RR cells. (A) By western blot analysis, the protein expression of Twist1 was examined in RR, RU and unsorted cells (labeled as 'Sox2R') from MCF7. MB231 was used as positive control. (B) MCF7-RR and -RU cells were treated with either scramble siRNA or Sox2 siRNA. By quantitative RT-PCR, the expression level of a panel of EMT/invasiveness inducers were examined, including Snail1, Slug, ZEB1, MMP2, MMP3, MMP9, Twist1, E-cadherin, N-cadherin, FAK and TGF-β. siRNA knockdown of Sox2 resulted in significant up-regulation of Twist1 and N-cadherin, down-regulation of E-cadherin in MCF7-RU cells but not -RR cells. No significant change was found in the expression level of Slug, Snail, and ZEB1, MMP2, MMP3, MMP9, FAK and TGF-β after siRNA knockdown of Sox2. Three representative results (i.e., Slug, Snail, and ZEB1) were shown. Scramble siRNA was used as a control. (C) By western blot analysis, the protein expression level of Twist1 was detected after siRNA knockdown of Sox2. (D) For the ChIP assay, a normal rabbit IgG antibody or a specific anti-Sox2 antibody was incubated with cross-linked chromatin extracted from MCF7-RR and -RU cells. Isolated DNA was amplified with primer designed against the proximal promoter of Twist1. Sox2 was found to bind to the gene promoter region of Twist1 only in RU but not RR cells. Input control that represents DNA isolated from chromatin before immunoprecipitation shows equal loading. n.s represents no significant difference.