PMC

Restin prevented EMT in breast cancer cells. (A) Morphologic changes in MDA-MB-231 cells upon Restin overexpression. Phase-contrast cell images were taken (×40 magnification). mRNA (B) and protein levels (C) of epithelial markers (E-cadherin and ZO-1) and mesenchymal markers (Fibronectin, N-cadherin and Vimentin) in MDA-MB-231 cells (Control and Restin overexpression) 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. Tubulin was used as a loading control. Results presented here were representative of three different experiments. (D) Morphologic changes in MCF-7 cells upon Restin knockdown plus TGF-β treatment. MCF-7 cells (si-Control and si-Restin) were seeded onto 6-well plates and TGF-β (5 ng/ml) were added for 6 days. Phase-contrast cell images were taken (×40 magnification). (E) Protein levels of epithelial markers (E-cadherin and ZO-1) and mesenchymal markers (Fibronectin, N-cadherin and Vimentin) in MCF-7 (si-Control and si-Restin) cells were determined by western blot. Tubulin was used as a loading control. Results presented here were representative of three different experiments. Restin: Restin overexpression; si-Restin: Restin knockdown.

Kaplan–Meier curves of overall survival (left panel) and recurrence/progression‐free survival (right panel) according to restin staining Haverage‐score (average H‐score in the triplicate of NSCLC tissues for each patient). Red lines: restin staining Haverage‐score <200. Blue lines: restin staining Haverage‐score ≥200. p, p‐value of the log‐rank test.

Restin expression evaluated by immunohistochemistry in 113 non‐small‐cell lung cancer (NSCLC) tissues. (a) Global view of three tissue microarrays (TMAs) consisting of NSCLC tissues coming from 113 patients and incubated with an antibody against restin (PA5‐13166). Scale bars: 5000 μm. (b–e) Representative images of NSCLC tumors with: (b) absent, (c) weak, (d) moderate, and (e) strong restin staining scores. Image magnification: 63x and 100x. Scale bars: 50 and 20 μm. Restin displayed a cytoplasmic staining, with nuclear enhancement (restin staining appears red, while tar spots appear brown).

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.