Fig. 7.

INF2 regulates traction force and ECM fibrillogenesis. (A, Left) Representative confocal images of EGFP-paxillin in mock-transfected control and MEF transfected with siRNAs targeting INF2 (INF2 KD) plated on fiducial-marked polyacrylamide substrates of stiffness, 8.6 kPa. (Scale bar, 10 µm.) Note the difference in size of the scale bar in control and INF2 KD cells. (Right) Bar graph of normalized traction force generated on substrates by control and INF2 KD cells as determined by Fourier transform traction microscopy (46). *P < 0.05, Student’s t test. n = 5 cells per condition. (B) Representative phase-contrast images of cell-derived matrix produced by mock-transfected control human foreskin fibroblasts (HFFs) or HFFs transfected with shRNA targeting INF2. White boxes indicate area zoomed below. [Scale bar, (Top) 400 µm; (Bottom) 100 µm.] (C) Representative confocal micrographs of Alexa-488 phalloidin staining of actin (green) with immunofluorescence of paxillin (red) and FN (blue) in control and INF2 KD MEF. White boxes indicate area zoomed below. White arrowheads indicate fibrils of FN near FA, red arrowhead indicates diffuse FN. [Scale bar, (Top) 20 µm; (Bottom) 5 µm.] Note the difference in size of the scale bar in control and INF2 KD cells. (D, Left) Representative confocal micrographs of control MEF, INF2 KD MEF, or INF2-KD MEF reexpressing INF2-GFP (rescue) transfected with mApple-actin (green) and plated on FN-coated coverslips. Images were acquired 6 h after Alexa-647–labeled FN (red) was added to the media and allowed to be incorporated into fibrils. White boxes indicate area zoomed below. White arrowheads indicate accumulation of labeled FN at the termini of stress fibers; red arrowhead indicates lack of FN accumulation. [Scale bar, (Left) 10 µm; (Right) 5 µm.] (Right) Graph of average labeled FN fluorescence under each cell normalized to cell area. n = 10 cells per condition, error bar = SD. *P < 0.05; N.S., not significant, Student’s t test.