Figure 8.

RhoG and PLEKHG6 are required for dorsal ruffle formation in A431 cells after EGF stimulation. A431 cells were transfected with control, RhoG-, or PLEKHG6-specific siRNAs. After 72 h, the cells were plated on fibronectin-coated coverslips (5 μg/ml). After 4 h of serum starvation, the cells were treated with 20 ng/ml EGF and DIC microscopy movies of individual cells were taken (10 s/frame; 480 s total). Cellular ruffling responses showed either a “dorsal ruffling phenotype” (A, top row, arrowheads indicate dorsal ruffles, also shown enlarged in the inset) or a “Rac-like ruffling phenotype” (A, bottom row). Bar, 20 μm. (B) Quantification of the occurrence of dorsal-ruffle phenotype versus Rac-like phenotype after transfecting the different siRNAs. Shown are averaged results from three independently performed experiments. Asterisks indicate significant difference compared with control siRNA transfected cells (Student's t test, p < 0.05 and p < 0.01, respectively). (C) HeLa cells were transfected with low amounts of expression constructs for mCherry or mCherry-PLEKHG6 and analyzed for dorsal ruffle formation after EGF treatment. Bars, 20 μm. (D) Quantification of the frequency of EGF induced dorsal ruffle formation of HeLa cells transfected with the indicated expression constructs. Shown are averaged results from three independently performed experiments. Asterisks indicate significant differences (Student's t test, p < 0.001).