Figure 7

Restin activated mir-200 transcription in a p73-dependent manner. (A) A schematic diagram of the human mir-200b/a/429-luciferase constructs (WT and its truncations) was shown. (B) A series of mir-200b/a/429 promoter activities were determined by luciferase reporter assay. Restin overexpression lentivirus were added into 293T cells and mir-200b/a/429 promoter truncation plasmids were transiently transfected. * p < 0.05 relative to Control lentivirus. (C) mir-200b/a/429 promoter (WT and p53 mutant) activities were measured by luciferase reporter assay. * p < 0.05 relative to Control lentivirus. (D) Western blot was performed to detect p53 and p73 levels in MCF-7, MDA-MB-231 and MDA-MB-157 cells. (E) Co-immunoprecipitation assay was performed to detect the exogenous interaction between Restin and p73. HERK-293 cells were transiently transfected with Flag-tagged Restin and His-tagged p73 plasmids. (Upper panel) Cell extracts were immunoprecipitated with mouse IgG or anti-His antibody and then blotted with anti-Flag antibodies. (Lower panel) Cell extracts were immunoprecipitated with mouse IgG or anti-Flag antibody and then blotted with anti-His antibody. (F) MDA-MB-231 stable cells (Control and Restin overexpression) were seed onto 24-well plates and co-transfected with Control or p73 siRNAs (si-Con and si-p73) and mir-200b/a/429 promoter construct. * p < 0.05 relative to si-Con group, # p < 0.05 relative to Control cells. (G) MDA-MB-231 stable cells (Control and Restin overexpression) were seed onto 6-well plates and transfected with control and p73 siRNAs (si-Con and si-p73). RT-PCR was performed to detect mir-200a and mir-200b expression levels. * p < 0.05 relative to si-Con group, # p < 0.05 relative to Control cells. (H) MDA-MB-231 stable cells (Control and Restin overexpression) were seed onto 6-well plates and transfected with control and p73 siRNAs (si-p73). Western blot was performed to detect ZEB1 and ZEB2 levels.