Generation of a double scar/wasp mutant. (A) SCAR and WASP localize as expected in SIKO cells. Top: GFP-SCAR localizes to pseudopods (arrows), whereas GFP-WASP is not found at leading edges in the presence of SCAR, and is restricted to puncta at the center and rear. Bottom: When SCAR is repressed, GFP-WASP relocalizes to pseudopods but not blebs (arrowheads; green: GFP-SCAR and GFP-WASP; red: RFP-LifeAct). Bar, 5 µm. (B and C) Loss of both SCAR and WASP from SIKO/wasp⁻ cells verified by Western blotting with anti-SCAR and anti-WASP antibodies. In each case, substantial repression of SCAR is seen between 48 and 60 h. SIKO/wasp⁻ cells are completely deleted for WASP; only SCAR is inducible. MCCC1 is the loading control (Davidson et al., 2013a). (C) Quantitation of several repeats normalized to the corresponding MCCC1 band, then to SIKO + DOX scar levels (n = 3 blots, one-way ANOVA, difference between SIKO and SIKO/wasA⁻ at 48 h not significant, P = 0.7135). (D) Loss of both SCAR and WASP causes growth arrest. Cells were grown in plates in axenic medium and counted every 12 h. SIKO/wasp⁻ cells were starved of DOX at time = 0 h. Around 60 h later, when SCAR expression is lost, growth completely ceases in SIKO/wasp⁻ cells but not controls lacking either scar or wasp alone (n = 3).