Skip to main content
. 2017 Mar 1;130(5):892–902. doi: 10.1242/jcs.196881

Fig. 1.

Fig. 1.

Vimentin depletion induces stress fiber assembly. (A,B) The intensities of Tpm4.2 and F-actin (as detected by fluorescent phalloidin) are increased in vimentin-knockout U2OS cells (A) and knockdown HDF cells generated using a vimentin siRNA pool (B). Panels on the left show representative images of control (arrowheads) and vimentin-depleted cells that were co-cultured on same plates. Panels on the right show the quantifications of normalized relative Tpm4.2 fluorescent intensities in control (A, 32 cells from nine images; B, 32 cells from nine images) and vimentin-knockout or knockdown cells (A, 38 cells from nine images; B, 33 cells from nine images). Mean intensity values of control and knockout or knockdown cells from each image were used for statistical analysis. ***P<0.001 (paired t-test). (C) Western blot analysis of actin and Tpm4.2 protein levels in control and vimentin-depleted U2OS (left panel) and HDF (right panel) cells. The blots were also probed with vimentin antibody to confirm that the vimentin-knockout U2OS cell culture is not contaminated by wild-type U2OS cells and to verify efficiency of vimentin depletion in siRNA-treated HDF cells, and with GADPH antibody to control equal sample loading. Molecular masses in kilodaltons (kDa) are indicated in the blots. (D) Quantification of the relative levels of actin (left panel) and Tpm4.2 (right panel) normalized to internal control GAPDH from five western blots. *P<0.05, ***P<0.001; N.S., not significant (paired t-test). (E) Vimentin-knockout results in increased cell contractility detected by traction force microscopy. Panels on the left show representative force maps of control and vimentin-knockout cells grown on 25 kPa polyacrylamide dishes with fluorescent nanobeads. The panel on the right shows the quantification of traction forces (root mean square traction) in control cells (n=47) and vimentin-knockout cells (n=47) from three independent experiments. *P<0.05 (Mann–Whitney–Wilcoxon rank-sum test). The data are presented as mean±s.e.m. Scale bars: 10 µm.