Fig. 6.

Overexpression of BIG1-C or BIG2-C reversed effects of BIG1 or BIG2 depletion on cell migration and stress fiber content. (A) After 72-h depletion of BIG1 or BIG2, cells were incubated for 24 h with EV, or BIG1- or BIG2-full length (F) or indicated fragments, before Transwell migration assays. Cells on undersides of filters after 4 h at 37 °C were fixed, stained with crystal violet, and counted as described in Materials and Methods. Data are means ± SEM of values from three experiments. *P < 0.005 vs. NT, **P < 0.01 vs. BIG1 siRNA + EV, ***P < 0.05 vs. BIG2 siRNA + EV. (B) Cells were incubated with vehicle (mock) or siRNA, either nonspecific (NT) or specific for BIG1 or BIG2, before transfection with indicated N-terminal HA- or GFP-tagged BIG1 or BIG2 constructs. After 24 h, cells were fixed, reacted with antibodies against HA, and stained for F-actin by Alexa Fluor-594-phalloidin before microscopy and imaging. (Scale bar, 10 µm.) (C) Box-blot analysis of integrated phalloidin intensity per cell area for each group (dotted line, mean; box, 25th, 50th, and 75th percentile; whiskers, maximum and minimum). Data for 40 cells from each of three experiments were analyzed. *P < 0.05, **P < 0.01, ***P < 0.001.