Fig. 3.
RSK2-stimulated cell migration requires Rho GTPase activity. (A and B) U87MG cells were transfected with Flag-EV or Flag-RSK2-Y707A together with HA-tagged DN-Rho isoforms or HA-EV and subjected to G418 selection and serum starved (24 h) before Transwell migration (A) or Matrigel invasion (B) assays. Results are presented as mean ± SD of four independent experiments each performed in triplicate. (C and D) U87MG cells infected with indicated rLenti-shRNA against different Rho isoforms were transfected with Flag-RSK2-Y707A. shLacZ infection and Flag-EV transfection were used as controls. Cells were subjected to G418 and puromycin selection and serum starved before Transwell assays. The results are presented as mean ± SD of three independent experiments performed in triplicate. (E–G) Lentivirus shRNA-mediated Rho silencing. U87MG cells expressing human WT-HA-Rho isoforms were transduced with the indicated lenti-shRNA against human RhoA (E), RhoB (F), or RhoC (G). Cells were subjected to puromycin selection before lysate preparation, and the levels of HA-Rho isoforms were detected by anti-HA immunoblotting. Actin was used as a loading control. For all figures, error bars represent SD. Statistical significance between the indicated sample versus EV+RSK2Y707A is *P < 0.05, **P < 0.01, or NS (not significant, P ≥ 0.05). Refer to Dataset S1 A and B for all P values.