Fig. 1

Design and validation of siRNA screen. a Schematic representation of the screen design and implementation. PC3 cells were stained with carboxyfluorescein succinimidyl ester (CFSE) and reverse transfected with siRNAs. Cells were then divided and seeded into one 96-well plate to carry out cell viability analysis and two separate 96-well plates to measure migration by Oris™ assay with and without hepatocyte growth factor (HGF) (Table 1). Hits from the screen were quantified. b Conditions for the migration screen were tested using an siRNA targeting Cdc42 (siCdc42-2, Additional file 1: Table S1) and control siRNA. Images show examples of CFSE-labelled PC3 cells immediately after removal of the Oris™ stopper (0 h) and 24 h later. Bottom images are thresholded 24 h images (see Methods). White dotted circle indicates wound area at 0 h. c. Quantification of the effect of Cdc42 depletion on migration in the Oris™ assay using the screen conditions, comparing cells on uncoated plastic and Matrigel-coated plastic. n = 3, mean +/− standard error of the mean (SEM); **p ≤ 0.01, *p ≤ 0.05, Student’s t test. d Single siRNAs targeting Cdc42 (siCdc42-2), RhoA (siRhoA-1) or control siRNA were tested for knockdown of protein expression using the screen conditions. Cells were lysed 72 h after transfection, and cell lysates western blotted with antibodies to Cdc42 and RhoA, and α-tubulin as a loading control