Hax1 depletion impairs uropod retraction and reduces RhoA activation. (A) Lentiviral knockdown of Hax1 (Hax1 shRNA A and B) in PLB-985 cells display significant depletion of Hax1 compared with control shRNA. Quantification was normalized to vinculin loading control. ***, P < 0.01; ANOVA with Tukey posttest. (B) Still image from a time-lapse video of control shRNA and Hax1 shRNA A and B PLB-985 cells during chemotaxis on 10 µg/ml fibrinogen (Videos 4 and 5). Arrowheads indicate elongated uropods of Hax1-deficient PLB-985 cells during chemotaxis. Bar, 100 µm. (C) Quantification of uropod length in Hax1-deficient cells compared with control. Control shRNA (n = 279) cells, Hax1 shRNA A (n = 275) cells, and Hax1 shRNA B (n = 241) cells were from three independent experiments. The graph represents means ± 95% confidence interval. ***, P < 0.01; one-way ANOVA with Tukey posttest. (D) GST-RBD pull-down assays show a 10-fold decrease in RhoA activity in Hax1-deficient cells compared with control. ***, P < 0.01; t test. (E) Control and Hax1-deficient PLB-985 cells expressing utrophin-GFP during chemotaxis to 1 µM fMLP on fibrinogen (Videos 6 and 7). For Rho-associated protein kinase inhibition, cells were incubated with 30 µM Y-27632 for 30 min before imaging. Fluorescence intensity from the front to the back of the cell was determined by line scan analysis (yellow lines). Bar, 10 µm. (F) Quantification of line scan analysis of utrophin-GFP localization from E. P-values were obtained by ANOVA with Tukey posttest. Red lines indicate the mean fluorescence ratio from three independent experiments. Error bars are means ± SEM from three independent experiments. A.U., arbitrary unit.