Figure 5

Restin downregulated ZEB1/2 expression at the post-transcriptional level. RNA (A) and protein levels (B) of FOXC2, Twist, ZEB1, ZEB2, Slug and Snail in Control and Restin overexpressed MDA-MB-231 cells were determined by real-time PCR and western blot. Values were expressed as means ± S.M. of at least three independent experiments. * p < 0.05 vs Control group. (C) Expression level of above proteins in si-Control and si-Restin MCF-7 cells treated with TGF-β was determined by western blot. Tubulin was used as a loading control. Results presented here were representative of three different experiments. (D) MCF-7 stable cells (si-Control and si-Restin) were seed onto 6-well plates and transfected with control and ZEB1 siRNAs (si-ZEB1). 48 h posttransfection, proteins were collected and western blot was performed to detect protein levels of E-cadherin, Fibronectin and Vimentin. Tubulin was used as a loading control. Result presented here was a representative of three different experiments. (E) ZEB1 promoter activity was determined by luciferase reporter assay. HEK293 cells were seeded in 24-well plates and transfected with Restin overexpression lentivirus and ZEB1 promoter plasmids. The firefly luciferase activity was normalized to that of the renilla luciferase. (F) ZEB1 3’UTR activity was determined in HEK293 cells by luciferase reporter assay upon Restin overexpression. HEK293 cells were seeded in 24-well plates and transfected with ZEB1 3’UTR plasmids and different dose of Restin overexpression lentivirus. The firefly luciferase activity was normalized to that of the renilla luciferase. Values were expressed as means ± S.M. of at least three independent experiments. * p < 0.05 relative to cells without addition of lentivirus (−), ^# p < 0.05 relative to Control group.