Fig. 6.

ZNF471 inhibits the expression of EMT markers. a Bright field microscopic images of SiHa cell morphology in the presence of EMT inducers. The expression of ZNF471 did not show any morphological changes except for TGF-β, while control cells showed mesenchymal phenotypes with all the EMT inducers. Control and ZNF471 expressing SiHa cells were treated with FGF-2 (5 ng/mL), TGF-β (2 ng/mL), IL-6 (25 ng/mL), EGF1 (50 ng/mL), and TNF-α (10 ng/mL) for 72 h to assess the EMT. b RT-PCR analysis of the basal level of epithelial and mesenchymal markers in control and ZNF471 expressing SiHa cells. The expression ZNF471 significantly up-regulated CDH1, inhibited VIM, CDH2, and TW1, and marginally reduced SNAI1, SNAI2, and CTNNB1 without changing ZEB1 and TW2 at mRNA level. c Bar graph representing the densitometry analysis of epithelial and mesenchymal marker levels. β-actin was used for normalization as an internal control in RT-PCR experiments. “*” indicates statistical significance (*P < 0.05). d Western blot analysis for EMT associated proteins. Presence of ZNF471 significantly downregulated mesenchymal markers (CDH2, VIM, ELK1, and RAF1) and EMT transcription factors (SNAI1, SNAI2) with the concomitant increase in the level of CDH1 in SiHa and CaSki cells. e Bar graphs showing normalized protein levels of EMT associated proteins in SiHa and CaSki cells. f Gel image of ChIP-PCR indicating direct binding of ZNF471 to VIM promoter