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. 2017 Feb 24;49(2):e296. doi: 10.1038/emm.2016.151

Figure 4.

Figure 4

Resveratrol (REV) inactivates RhoA. (a) The serum-starved MDA-MB-231 cells were pretreated with REV (25 μM) and then stimulated with lysophosphatidic acid (LPA; 5 μM) for 3 min. Active RhoA bands were quantified by ImageJ densitometric analysis and normalized to total RhoA (**P<0.01 versus control, #P<0.05 versus LPA treatment). (b–e) The MDA-MB-231 cells were transfected with the indicated vectors for 48 h and then treated with REV (25 μM) for 1 h. (b) RhoA-GTP pull-down assays. Active RhoA bands were quantified by ImageJ densitometric analysis and normalized to total RhoA (**P<0.01 versus control, ##P<0.05 versus transfection of RhoAV14). (c) Immunoblotting. Immunoblot bands were quantified by ImageJ densitometric analysis and normalized to the control (*P<0.05 versus control vector, **P<0.01 versus control vector, ***P<0.001 versus control vector, #P<0.05 versus transfection of RhoAV14, ##P<0.01 versus transfection of RhoAV14, ###P<0.001 versus transfection of RhoAV14). (d) In vitro invasion assay (***P<0.001 versus control vector, ###P<0.001 versus transfection of RhoAV14). (e) RhoA GLISA activation assay (*P<0.05 versus control, ##P<0.01 versus V14 RhoA treatment only). (f–h) The serum-starved MDA-MB-231 cells were pretreated with simvastatin (5 μM) for 1 h and then stimulated with LPA (5 μM) for 2 h. (f) Immunoblotting. Immunoblot bands were quantified by ImageJ densitometric analysis and normalized to Lats1 and YAP (*P<0.05, **P<0.01 versus control, ##P<0.01 versus LPA treatment only). (g) The expression of YAP was visualized by immunofluorescence. Original magnification × 200; scale bar, 50 μm. (h) In vitro invasion assay (***P<0.001 versus control, ###P<0.001 versus LPA treatment only). (i) The serum-starved MDA-MB-231 cells were pretreated with REV (25 μM) or simvastatin (5 μM) for 1 h and in vitro invasion was analyzed against LPA (5 μM) or epidermal growth factor (EGF; 100 ng ml−1; ***P<0.001 versus control, ###P<0.001 versus LPA or EGF treatment only, $$$P<0.001 versus LPA or EGF and simvastatin treatment). All experiments were repeated three times.