WASP is crucial for pseudopod formation of neutrophils. (A) Western blots showing WASP and WAVE2 expression in control HL-60 cells, cells expressing shRNA to WASP, and exogenous WASP with three silent mutations in the region corresponding to the shRNA (KD + rescue) or cells expressing anti-WASP shRNA and empty vector rescue control (KD + control). Approximately equal amounts of total protein were loaded in each lane, which was confirmed by using actin as a loading control. (B) Brightfield images of control and WASP-KD cells. (C) KD of WASP using shRNA reduces the percentage of cells that have pseudopods. (D) Immunofluorescence of control and WASP-KD HL-60 cells showing microtubules (green, antibody stained), actin filaments (red, phalloidin stained), and DNA (blue, DAPI). Note the signature rhino phenotype in the WASP-KD cell. See also Fig. S2. (E) Rescue of rhino protrusion phenotype by expression of shRNA-insensitive WASP as described in A. Note: no wild-type (WT) cells were observed to exhibit the rhino phenotype. (C and E) Means and SD (bars) of three biological replicates (dots) with >130 cells total; p-values were obtained with two-tailed paired t tests. (F) Maximum projections of spinning-disk confocal stacks of living HL-60 cells with fluorescent probes specific for polymerized actin (mCherry fused to the calponin homology domain of Utrophin; Utr261). Insets are cross sections through the rhino horns at positions indicated by yellow lines, confirming that they are hollow with a shell of actin. See also Fig. S2 for a time lapse of the right-hand cell showing the dynamics of the rhino horn protrusion.