Rac-dependent RhoH effects on cortical F-actin assembly and chemotaxis. (A,B) Levels of the active, GTP-bound Rac proteins of Lin⁻/c-kit⁺ cells after stimulation with 100 ng/mL SDF-1α. The levels of GTP-bound Rac and total Rac proteins in whole-cell lysates were examined in parallel. Ratio of GTP-bound Rac GTPases was quantified by densitometry measurements. The relative ratio is shown as the mean plus or minus SD (n > 3) except WT 5-minute data (average±range; n = 2). (C) Expression of EGFP–Rac1N17 inhibited chemotaxis induced by SDF-1α. WT, Rhoh^−/− LDBM cells were transduced with retroviral viruses expressing EGFP-tagged the dominant-negative Rac1 mutant Rac1N17 (EGFP–Rac1N17) or vector control. The percentage of sorted EGFP⁺/c-kit⁺ cells migrated toward SDF-1α in a transwell chamber for 4 hours was calculated. Data represent the mean plus or minus SD; n = 3. (D) Inhibitory effect of EGFP–Rac1N17 on cortical F-actin assembly. Sorted EGFP⁺/c-Kit⁺ cells were stimulated with SDF-1α (100 ng/mL) for 30 seconds and stained with rhodamine-labeled phalloidin and 4′,6-diamidino-2-phenylindole (DAPI). A total of 200 cells were counted under fluorescent microscope. Data represent the percentage of cells with cortical F-actin as the mean plus or minus SD; n = 3.