Fig. 6

CSRP2BP regulates N-cadherin transcription by recruiting SMAD4 in cervical cancer. A The interaction between CSRP2BP and SMAD4 was tested by Co-IP assay with anti-Flag antibody and control normal IgG. Western blotting was performed with an anti-HA antibody. B Subcellular co-expression of CSRP2BP (red) and SMAD4 (green) in Hela cells was detected by immunofluorescence staining. Nuclei were counterstained with DAPI (blue). (Scale bar, 20 µm). C A luciferase reporter gene assay was used to evaluate SMAD4 promoting N-cadherin promoter transcription (n = 3). D Position of the SMAD4-binding site in the N-cadherin promoter. E CSRP2BP occupies the E-box, SBE1 and SBE2 of the N-cadherin promoter region, as measured by ChIP-PCR assay. F CSRP2BP levels in the N-cadherin promoter region were analysed by ChIP‒QPCR assay. IgG was used as a negative control. G The wild type and mutated constructs of the N-cadherin promoter-luciferase reporter (N-cadherin-Luc). SEBM-Luc had mutated SEB2. H Cell transfection assays. CSRP2BP/SMAD4 complex showed much less increased the activities of SBEM-Luc versus N-cadherin-Luc in Hela cells. I Hela-CSRP2BP cells and control cells were pre-treated with SMAD4 siRNA for 48 h and then detected by Western blotting. J Wound healing assays and Transwell assays K revealed that the migration and invasion rates decreased in Hela-CSRP2BP-siSMAD4 cells. Data are shown as the mean ± SD (n = 6). L Hela-CSRP2BP cells and control cells were pretreated with SMAD4 siRNA for 48 h and then detected SMAD4 expression by Western blotting. M CSRP2BP and H4Ac levels in the N-cadherin promoter region were analysed by ChIP‒QPCR assay upon SMAD4 knockdown in Hela-CSRP2BP cells. IgG was used as a negative control. (*P < 0.05, **P < 0.01, ***P < 0.001, ****P<0.0001)