Smurf1 inhibits focal adhesion formation. (A) Indicated plasmids were transfected into HeLa cells, then the cells were plated on FN-coated coverslips for 30 or 60 min and immunoreacted with anti-Paxillin and anti–Kindlin-2 or Flag antibodies. Expression and localization of GFP-Smurf1, Kindlin-2, and paxillin were observed under a confocal microscope with 63× objective. Bars, 5 µm. (B and C) Numbers of paxillin-staining focal adhesions per cell (B) and unit area (C) were quantified. Values are mean ± SD of three independent experiments. **, P < 0.01 vs. GFP + Flag group; ##, P < 0.01 vs. GFP + Flag-Kindlin-2 group. *, P < 0.05 vs. GFP + Flag group; #, P < 0.05 vs. GFP + Flag-Kindlin-2 group. Smurf1CA, Smurf1 C699A. (D–F) WT, Smurf1^−/−, or Smurf1 rescue MEFs were plated on FN-coated coverslips for 30 min and immunoreacted with antibody to Kindlin-2 and Paxillin. Expression of Kindlin-2 and Paxillin was determined by confocal microscopy under 63× objective. Bars, 5 µm. Numbers of paxillin-staining focal adhesions per cell and unit area were quantified (E and F). Values are mean ± SD of three independent experiments. *, P < 0.05 vs. WT group; #, P < 0.05 vs. Smurf1 KO group. (G–J) HeLa cells expressing EGFP-Zyxin were transfected with control siRNA or Smurf1 siRNA, plated on FN until fully spread (5 µg/ml), and then analyzed using time-lapse confocal microscopy with 100× objective. Bars, 10 µm. (G) Rate (per minute) of assembly (H), disassembly (I), and lifespan (J) of FAs as measured by change in GFP fluorescence over time for control siRNA and Smurf1 siRNA cells. Values are mean ± SD of three independent experiments; *, P < 0.05 vs. control group.