Figure S5 from RASSF1A Directly Antagonizes RhoA Activity through the Assembly of a Smurf1-Mediated Destruction Complex to Suppress Tumorigenesis

Figure S5. RASSF1A inhibits tumor-promoting activity of RhoA. A, RASSF1A regulation of cyclins and CDK inhibitors in a RhoA-dependent manner. B, Blockade of RhoA repression of p21WAF1 expression by WT-RASSF1A but not by RASSF1A-Δ256-277 and RASSF1A-Δ69-82. C, Inhibitory effect of WT-RASSF1A but not of RASSF1A-Δ256-277 and RASSF1A-Δ69-82 on EGF-induced DNA synthesis. DNA synthesis was measured by (3H)thymidine uptake assay after 24 h treatment. Data represent the mean {plus minus} SD (n = 3 experimental replicates; **, P < 0.01, Student t test). D, Flow cytometric assay of sub-G1 fraction showing no apoptotic activity of RASSF1A-R269F. E, RASSF1A inhibition of cell migration in a RhoA-dependent manner. Cell migration was compared at 18 h after artificial scratch. F, Matrigel assays showing blockade of EGF-induced tumor cell invasion by WT-RASSF1A but not by RhoA binding-deficient mutants (R269F and L266G). Data represent the mean {plus minus} SD (**, P < 0.01, Student t test). G, Time- and dose-dependent RhoA activation by TGF-β1 in DU145 cells. GTP-RhoA level was determined by GTPase pull-down assay using Rhotekin RBD-agarose. H, A crucial role for RhoA in TGF-β1-induced EMT in DU145 cells. The cells were transfected with siRhoA, siCdc42 or siRac1 and treated with TGF-β1(2 ng/ml, 48 h). I, Effect of RASSF1A or Smurf1 depletion on TGF-β1-induced EMT. HeLa and HaCaT cells transfected with siRASSF1A or siSmurf1 were treated with TGF-β1 (2 ng/ml, 48 h). TGF-β1-induced EMT was evaluated by microscopic analysis.