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Fig. 4. Effect of p190 knockdown on podosome formation and function. (A) p190A and p190B localised to podosomes in HUVECs. F-actin was labelled using rhodamine-phalloidin and p190 isoforms were labelled using anti-isoform-specific antibodies and secondary Alexa-Fluor-488-conjugated antibodies in HUVECs treated with PMA. Arrows indicate podosome structures. Scale bar: 10 µm. (B) Knockdown of p190B did not alter podosome formation, but knockdown of p190A increased the number of cells showing podosomes. HUVECs were transfected with siRNA designed against p190A or p190B for 65 hours, treated for 1 hour with PMA and stained for F-actin. Cells showing podosomes were counted and data are presented as a percentage compared with the control (control-siRNA-transfected cells). Error bars represent the mean±s.e. of ten (A1 and B1) or three (A2 and B3) independent experiments. *P<0.0005; #P<0.05 in Student's t-test when compared with control. (C) Alteration of in situ proteolytic activity in p190-KD cells. HUVECs transfected with the indicated siRNA were seeded on FITC-gelatin-coated coverslips. Half of the coverslips were treated with PMA for 1 hour to induce podosome formation. Cells were fixed and stained for F-actin. `Black holes' representative of gelatin-degradation zones were recorded as described in the Materials and Methods section and are presented as a percentage of the control response obtained with untreated cells. Each bar represents the mean±s.e. of four independent experiments. The effects obtained were statistically significant (#P<0.05; *P<0.01 in Student's t-test) for p190A- and p190B-KD cells.
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