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. 2017 Sep 16;8(50):87364–87378. doi: 10.18632/oncotarget.20957

Figure 4. Knockdown of RhoC but not RhoA precludes the pro-invasive effect of CNF1- and CNFY treatment.

Figure 4

(A) Left: Western-blots showing the knockdown efficiency for six constitutively expressed shRNA constructs against RhoA (shRhoA, top) and RhoC (shRhoC, bottom) plus a non-silencing control construct (NS) in WT-MCF-10A cells. Right, corresponding statistical analysis of the three most effective constructs (N = 4). Although not the most effective, shRhoA_3 was chosen for knockdown of RhoA in further experiments, because it was already used before [58]. (B) Phase-contrast micrographs of MCF-10A 3D cultures (DI3D7) transfected with the non-silencing (NS, top), shRhoA (middle) or shRhoC constructs (bottom). Cultures were grown in untreated control conditions (left) or treated with 1 nM CNF1 (middle) or CNFY (right) during DI3D1-7. Transfection with shRhoC but not shRhoA antagonizes the pro-invasive effect of CNF1/Y treatment. Scale bar 50 μm. (C) Quantification of the functional invasiveness by the Xcelligence® device. MCF-10A cells transfected with NS, shRhoA or shRhoC were analyzed under untreated control conditions (white bars) or treated with either 1 nM CNF1 (black bars) or 1 nM CNFY (grey bars). Bars and error bars represent mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; two-tailed, one-sample t-test and one-way ANOVA.