Fig. 6. PMA-induced podosomes depend on RhoGTPases in HUVECs. (A) V12Cdc42-induced podosomes are insensitive to PKC inhibitors. HUVECs were transfected with a GFP-V12Cdc42 construct, and treated with Gö6976 and rottlerin. F-actin was labelled with rhodamine-phalloidin. Arrows indicate cells expressing the GFP-tagged constructs (visualised by the green GFP signal). The percentage of cells transfected with V12Cdc42 that show podosomes is indicated at the top of each image. Bars, 10 µm. (B) siRNA designed against RhoA, Rac1 or Cdc42 downregulated their respective target protein. HUVECs were transfected with the indicated siRNA. After 48 hours, protein extracts were analysed by western blot. (C) Knockdown of Cdc42 or RhoA expression inhibited PMA-induced podosome formation. HUVECs were transfected with indicated siRNA and analysed by immunofluorescence. Quantitation of the experiment was done by counting the number of cells showing podosomes after transfection and is presented as percentage of the control response obtained with cells transfected with control siRNA. Error bars represent the mean ± s.d. of two independent experiments.