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. 2019 Sep 22;20(19):4697. doi: 10.3390/ijms20194697

Figure 5.

Figure 5

Reduction of RhoE upregulates RhoA activity during TGF-β-mediated EMT in HeLa cells. (A) Whole-cell lysates prepared from HeLa cells treated with TGF-β1 or vehicle were used for the GST pull-down assay with GST or GST–rhotekin Rho binding domain (RBD). The lower panel indicates Coomassie blue staining of GST proteins. Bound protein samples were immunoblotted with anti-RhoA antibody. The input volume was 5% of that of the cell lysates for the pull-down assay. The ratio of GTP-RhoA/total RhoA was quantified. The immunoblot shows the representative result and data in each column represent the mean and standard deviation of three independent experiments. The statistical significance of differences between two groups was evaluated using a two-tailed Student’s t-test. ** p < 0.05. (B) HeLa cells were transfected with siRhoE-A or a control siRNA and treated with 1 ng/mL TGF-β1 for 72 h. Whole-cell lysates were used for the GST pull-down assay with GST–rhotekin RBD. Bound protein samples were immunoblotted using anti-RhoA antibody. The input volume was 5% of that of the cell lysates for the pull-down assay. The ratio of GTP-RhoA/total RhoA was quantified. The immunoblot shows the representative result and data in each column represent the mean and standard deviation of three independent experiments. The statistical significance of differences between two groups was evaluated using a two-tailed Student’s t-test. * p < 0.01. (C) Protein expression of fibronectin and snail in control and RhoE knockdown cells treated with Y27632. HeLa cells were transfected with siRhoE-A or a control siRNA and pretreated with or without 10 μM Y27632 for 2 h followed by stimulation with vehicle or TGF-β1 for 72 h. Whole-cell lysates were subjected to Western blot analysis and β-actin was used as a loading control. Signal intensities of proteins were quantified using NIH-Image software. Immunoblots show representative results and each column represents the mean and standard deviation of three independent experiments. Statistical significance was assessed using one-way ANOVA with a post hoc Tukey–Kramer HSD test. * p < 0.01 and ** p < 0.05.