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. 2011 Dec;31(23):4789–4800. doi: 10.1128/MCB.05583-11

Fig. 6.

Fig. 6.

Knockdown of endogenous RhoGAP22 levels causes defects in insulin-regulated morphological changes. (A) Knockdown of RhoGAP22 in L6 myoblasts using siRNA. L6 myoblasts were transfected with 20 pmol of nontargeting control siRNA (Scr), 20 pmol of siRNA directed against an unrelated protein (TBC1D4), or either 20 pmol of RhoGAP22 siRNAs individually (1, 2, and 3) or 6.66 pmol of each RhoGAP22 siRNA pooled (1+2+3). Cells were cultured for 48 h prior to lysis and Western blotting with indicated antibodies. (B) RhoGAP22 knockdown caused defects in insulin-regulated morphological change. L6 myoblasts were transfected with either control siRNA (Scr) or pooled RhoGAP22 siRNAs (Kd). Cells were cultured for 48 h prior to serum starvation and treatment (Ins) or no treatment (Basal) with 100 nM insulin for 20 min. Cells were fixed and the actin cytoskeleton stained with Alexa 647 phalloidin. Representative images are shown. (C) Quantitation of defective morphological change in RhoGAP22 knockdown cells. Micrographs from panel B were analyzed using the Cell Profiler software package. At least 15 cells were analyzed in each condition. Error bars indicate standard errors of the means. Significance was determined by one-way ANOVA followed by Tukey's posttest. *, P < 0.05; ***, P < 0.001.