Fig. 5. Knockdown of endogenous l-caldesmon by siRNA enhances podosome formation. (A,B) A7r5 cells were transfected with the caldesmon-specific siRNA1120 (1120) and siRNA143 (143) or with a negative control siRNA (control) for 48 hours prior to lysis and western blot analysis. Samples were probed with a general l-caldesmon antibody (l-Cad) or ß-actin as a loading control. (A) Blot is representative of three independent experiments. (B) Densitometry was performed to quantify the level of knockdown by each caldesmon siRNA compared with the negative siRNA control, as described in the Materials and Methods. The caldesmon siRNA143 and siRNA1120 reduced the level of caldesmon in the total population of transfected and untransfected cells by 20% and 40%, respectively. This translates to about 40% and 80% knockdown of caldesmon expression, respectively, by siRNA143 and siRNA1120, taking into account of 50% transfection efficiency. (C-E) A7r5 cells co-transfected with siRNA1120 and EGFP-ß-actin were fixed and stained 48 hours after transfection (C) or stimulated with 1 µM PDBu for 30 minutes prior to fixation (D,E). Cells were stained for EGFP (green) to identify siRNA transfected cells and l-caldesmon (red). An overlay of the two channels is shown. (C) Cells transfected with siRNA1120 show a significant decrease in the level of caldesmon but still retain stress fibres. (D,E) Stimulation of cells in which the level of caldesmon has been reduced by siRNA1120 with PDBu show an increase in both the number of cells displaying podosomes and the number of podosomes per cell. Bars, 20 µm in C-E.