Fig. 2. VEGF plays a critical role in the activation and localisation of Cdc42.

a CRC cells were starved overnight in 1% FBS and then treated with either VEGF or not. Lysates were obtained, and Cdc42-GTP levels were assessed using the PAK1 pull-down assay. b Cell membrane fractions were isolated and probed for Cdc42. The NaK probe is a reference for membrane-bound proteins. c Cdc42/CD44 staining of SW480/LS174T cells. SW480/LS174T cells were treated as follows: SW480/LS174T cells were starved overnight in 1% FBS (VEGF-0 h); SW480/LS174T cells were starved overnight in 1% FBS and treated with 50 ng/mL VEGF for 6 h (VEGF-6 h). CD44 is a reference protein for the cell membrane. Scale bar, 5 μm. d Representative confocal image of CRC cells showing Cdc42 imaged with EGFP. Cdc42 protein expression was measured using western blotting, and Cdc42 was amplified using real-time quantitative polymerase chain reaction. Error bars represent the mean ± SD of triplicate experiments; **P < 0.01, *P < 0.05. Scale bar, 100 μm. e, f CRC cells were cultured to confluency on glass coverslips and then starved, scratched, and treated with VEGF. As shown in e, Cdc42 is located at the front of live CRC cells. Scale bar, 10 μm. Frames from typical time-lapse sequences are presented. Arrowheads denote the recognisable front of the cells at 15 min in SW480 cells and at 9 min in LS174T cells. Fixed Cdc42 cells were subjected to fluorescent immunostaining as shown in f (in red), and the nuclei were stained blue with [4′,6-diamidino-2′-phenylindole (DAPI). White arrows point to Cdc42 expression in cells oriented towards the scratched edge. The white reference line is parallel to the scratched edge and determines individual cell polarisation vs. the wound. Scale bar, 20 μm. g Quantitative analysis showed the cell percentages with Cdc42 expression oriented in the front of the cells; error bars represent the mean ± SD of triplicate experiments, *P < 0.001.