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. 2014 Mar 7;44(5):1499–1511. doi: 10.3892/ijo.2014.2330

Figure 5.

Figure 5.

Constitutive activation of RhoA inhibits cell motility. (A) MDA-MB-231 cells were left untransfected or transfected with a dominant active RhoA construct (RhoA DA). Cells were grown in a monolayer then wounded and left to recover the wound then imaged at the same frame after 16 h (lower micrographs). Wound widths were measured at 11 different points for each wound, and the average rate of wound closure was calculated in μm/hr. Data are the mean ± SEM from 3 wound healing assays from 3 independent experiments. The results were significant with ***p<0.001. Right panel, cells were transfected as described, lysed and immunoblotted for RhoA. The gels were quantitated and data are the mean ± SEM from 3 different gels. (B) Western blot showing the decrease in Rac1 expression compared to actin (lower panel) in cells transfected with Rac1 siRNA compared to luciferase control (left lane). MDA-MB-231 cells were grown in a monolayer then wounded and left to recover the wound then imaged at the same frame after 16 h (lower micrographs). Quantitation of wound widths were measured at 11 different points for each wound. (C) The average rate of wound closure for the luciferase and the Rac1 siRNA-transfected cells was calculated in μm/hr. Data are the mean ± SEM from 3 wound closure assays from 3 independent experiments. The results were significant with **p<0.01. (D) MDA-MB-231 cells were transfected with both StarD13-siRNA and RhoA-siRNA double knockdown or both StarD13-siRNA and Rac1-DA construct. Cells were grown in a monolayer then wounded and left to recover the wound then imaged at the same frame after 16 h. (E) The average rate of wound closure for the control and transfected cells was calculated in μm/hr. Data are the mean ± SEM from 3 wound closure assays from 3 independent experiments. The results were significant for the StarD13+RhoA-KD with **p<0.002 and not significant (ns) for StarD13KD+RacDA. (F) The percentage of wound area at 16 h following wounding. Data are the mean ± SEM from 3 wound closure assays from 3 independent experiments. The results were significant for the StarD13+RhoA-KD with **p<0.002 and not significant (ns) for StarD13KD+RacDA.