Figure 6. Kindlin-1 forms a complex with SARA, TβRI and Smad3 to activate TGF-β/Smad3 signaling in CRC cells.

A. Kindlin-1 interacts with SARA in CRC cells. HCT116 cells were transfected with Flag or Flag-Kindlin-1 expression vector for 48 h, later proteins were extracted for co-IP assays using an anti-Flag antibody, followed by Western blot analysis using indicated antibodies. B. Association of SARA, Kindlin-1, Smad3 and TβRI in living CRC cells. Proteins were extracted from HCT116 cells and co-IP assays were performed using anti-TβRI (Left panel) or anti-Smad3 (Right panel) antibodies, followed by Western blot analysis using indicated antibodies. C. Kindlin-1 regulates interaction between Kindlin-1 and TβRI in CRC cells. HCT116 cell lysates with Kindlin-1 overexpression or Kindlin-1 knockdown were co-immunoprecipitated using an anti-Smad3 antibody, controlled by empty vector or control siRNA. Western blot assays were performed using indicated antibodies. D. Upper panel: Kindlin-1 rescues SARA-depletion caused Smad3 inactivation. Control or SARA siRNA was transfected into HCT116 cells with or without Kindlin-1 overexpression. Western blot assay was performed using indicated antibodies. Lower panel: SARA rescues Kindlin-1-depletion caused Smad3 inactivation. Control or Kindlin-1 siRNA was transfected into HCT116 cells with or without SARA overexpression. Western blot assay was performed using indicated antibodies. E. Kindlin-1 is required for TGF-β-induced CRC cell migration. Control or Kindlin-1 siRNA was transfected into HCT116 cells. Twenty-four hours later cells were treated with TGF-β1 for another 12 h, then cell migration assay was performed. Data were expressed as mean ± SEM from three independent experiments, p<0.01.