Fig. 2

Screen of Rho RNAi library identifies four cell migration phenotypes. The migration screen was performed with PC3 cells transfected with siRNA pools or single siRNAs targeting the indicated genes. After 24 h, cells were seeded at confluence into 96-well plates containing Oris™ stoppers. After 24 h, the stoppers were removed and cells were treated with or without 10 ng/ml HGF for 24 h. Images of cells were acquired immediately after removing the stoppers (0 h) and 24 h later. a Graph shows migration Z-scores (see Methods) for the GTPases included in the screen in normal medium (top panel) or medium containing HGF (bottom panel); n = 3. b Representative images of migration area (24 h after stopper removal) for representative hits from each phenotypic category: no effect on migration (siControl), accelerated migration (RhoQ, Rac3), impaired migration (RhoC, RhoV), impaired migration only in response to HGF (RhoBTB3). White dotted circle indicates wound area at 0 h. c Pie charts show number of genes giving each phenotype, based on type of gene (Rho GTPase, RhoGEF, RhoGAP, Rho effector). Note that BCR, which contains both a RhoGEF and a RhoGAP domain, was included as a GAP in the pie chart for accelerated migration and indicated with an asterisk. d Migration for cells transfected with the indicated single siRNAs is shown as percentage of control siRNA-transfected cells +/− SEM, n = 3; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, Student’s t test