JNK is activated downstream of G12 signaling in 4T1 mammary carcinoma cells and is required for cell invasion.

<p>(A) 4T1 cells were infected with adenovirus harboring GFP (control) or the RGS domain of p115RhoGEF (p115RGS) for ∼20 h, and then starved for 8 h. Cells were allowed to invade Matrigel for 24 h in the presence of thrombin (1 U/ml), towards 3% FBS. Cells that invaded were stained and counted in four random optical fields for each transwell, with three transwells per condition. Results are shown as percent invasion compared to invasion in the control cells. The <i>inset</i> shows p115RGS expression. Data is presented as mean ± SE from a single experiment that is representative of three independent experiments. * <i>p</i><0.05 as determined by one-way ANOVA, followed by Tukey's multiple comparison test to obtain <i>p</i> values. (B) 4T1 cells were infected with adenovirus harboring GFP (control) or p115RGS, as indicated, for ∼18–19 h and starved for ∼9 h. Cells were then treated with 1 U/ml thrombin for 10 min, as indicated, before lysis in JNK activation buffer. Equal amounts of total protein in the lysates were separated by SDS-PAGE and immunoblot analysis was performed to assess levels of phospho-JNK (P-JNK, p54 and p46) and phospho-c-Jun (P-c-Jun). Total levels of JNK, myc-p115RGS and RhoGDI (loading control) are also shown. Data shown are from a single experiment that is representative of three independent experiments. (C) 4T1 cells were transfected with control (luc) and JNK siRNA for 67–68 h, and then starved for 8 h. Cells were allowed to invade Matrigel for 24 h in the presence of thrombin, towards 3% FBS. Cells that invaded were quantified as detailed for panel A. Results are shown as percent invasion compared to invasion in the control cells. The <i>inset</i> shows immunoblot analysis of JNK and Rho GDI (loading control) levels. Data is presented as mean ± SE from a single experiment that is representative of three independent experiments. ** <i>p</i><0.01 as determined by one-way ANOVA, followed by Tukey's multiple comparison test.