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Fig. 6. SCAR RNAi in BG2 cells inhibits lamellipodia and filopodia. (A) Immunoblotting. Extracts of BG2 cells subjected to SCAR RNAi for 4 days were probed with antibodies to SCAR and WASp. Reduction of SCAR is 90-95%. WASp is not changed. (B) Morphology and actin distribution. Overall cell shape shows decreased spreading and formation of long narrow processes. Phalloidin staining reveals decreased amount of actin, especially at the periphery. (C) Morphometry. Projected area (left) and P/A^1/2 cell shape index (right) of control (gray bars, n=14) and BG2 cells treated with dsSCAR for 4 days (black bars, n=18). (D) Cytoskeleton organization of SCAR-depleted BG2 cells (EM). Left: overview; neither normal lamellipodia nor filopodia are seen. Right: enlarged is boxed region from left panel; cell processes contain central actin filament bundles (arrowheads) and distal patches of the dendritic network (arrows). (E) Filopodia formation during spreading of control (upper row) or SCAR-depleted for 4 days (lower row) BG2 cells. Time-lapse sequences were acquired 2 hours after cell re-plating. Newly formed filopodia are indicated by arrowheads. Time in seconds. (F) Rate of filopodia initiation. Number of de novo formed filopodia per 2 minutes interval in control cells (gray bar, n=14) or cells treated with dsRNA (black bars, n=15) specific for SCAR (dsS) or WASp (dsW). SCAR-depleted BG2 cells have a significantly reduced ability to form filopodia. Bars, 10 µm (B), 1 µm (D) and 5 µm (E).

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