Fig. 4. PMA-induced podosomes depend on PKC and PKC activation. (A) Both the isotype-restricted and the isotype-selective inhibitor prevented PMA-induced podosomes in HUVECs. Cells were pretreated with either 50 nM Gö6976 or 3 µM rottlerin for 1 hour and then activated with PMA for 1 hour in the presence of the inhibitor. Cells were labelled for F-actin. Bar, 10 µm. (B) Quantitation of the experiment described in A. Cells showing podosome-like structures were counted after 1 hour treatment with PMA. Error bars represents the mean ± s.d. of three independent experiments. (C) siRNA designed against PKC and PKC downregulated their respective target protein. HUVECs were transfected with PKC or PKC siRNA and protein extracts were analysed by western blot. (D) Knockdown of PKC or PKC expression inhibited PMA-induced podosome formation. HUVECs were transfected with siRNA designed against PKC or PKC. Cells showing podosomes after 1 hour of PMA treatment were counted and data are presented as percentage of the total population. Error bars represent the mean ± s.d. of three independent experiments.