DOCK8 signals through CDC42 and PAK to maintain shape integrity. (A) Top: Proportions of T cells elongated during migration in collagen gel matrices. Normal human T cells were transfected with nonspecific (NS), DOCK8, CDC42, or RAC1/2 siRNA. Bottom: representative nuclear morphology of these cells migrating in collagen gels. BF, brightfield. DAPI, blue. (B) Proportions of dead or dying cells were quantitated by flow cytometric analysis of Annexin V and PI staining, after cells from A were allowed to migrate in collagen gels or in medium. (C) Knockdown efficiencies for A and B. Quantitative real-time RT-PCR to measure transcript levels of DOCK8, CDC42, RAC1, and RAC2, normalized to β-actin. Knockdown efficiencies of these genes were calculated as normalized levels in T cells transfected with indicated siRNA, divided by normalized levels in T cells transfected with nonspecific siRNA. Ratios are means ± SD of five independent experiments. (D and E) Proportions elongated within collagen gels of normal human T cells either treated with DMSO or the PAK inhibitor IPA3 (D), or transfected with NS or PAK1/2 siRNA (E). (F) Similar analysis as in B, using cells from E. Representative immunoblot showing efficiency of PAK1/2 knockdown. (G) Morphology of T cells in which WAS gene expression was silenced, during migration in collagen gel. (H) Proportions of Annexin V⁺ or PI⁺ cells, after migrating in collagen gel matrices or medium. Normal human T cells were transfected with NS, WAS, or DOCK8 siRNA. Representative immunoblot showing WASP knockdown efficiency in human T cells. Bars, 10 µm. A, B, D, E, G, and H are representative of three independent experiments, and F of five experiments. A–F and H show means ± SD. One-way ANOVA was performed for A, two-way ANOVA for B, F, and H, and unpaired two-tailed Student’s t test for D and E. Statistical significance indicated by *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, nonsignificant.