RhoH regulates subcellular localization of Rac1. (A) WT LDBM cells were transduced with retroviral vectors expressing HA-tagged RhoH-IRES-YFP (RhoH) or vector control (YFP). Sorted YFP⁺/c-Kit⁺ cells were stimulated with SDF-1α (100 ng/mL) for 30 seconds, fixed, and stained with anti-Rac1 mAb (green), rhodamine-labeled phalloidin (red) and 4′,6-diamidino-2-phenylindole (DAPI; blue). Phase contrast images show the cellular morphology. Some cells were polarized after SDF-1α stimulation. The leading edge of polarized cell is indicated with a white arrow. (B) EGFP-Rac1, EGFP-Rac1V12, and EGFP-Rac2 localization in WT and RhoH-overexpressing cells. WT LDBM cells were transduced with retroviral vectors expressing Rac1 (EGFP-Rac1), EGFP-tagged Rac2 (EGFP-Rac2), or constitutive active Rac1 mutant Rac1V12 (EGFP-Rac1V12) with or without HA-tagged RhoH-IRES-YFP (RhoH–YFP). Sorted EGFP⁺/c-Kit⁺ or EGFP⁺/YFP⁺/c-Kit⁺ cells were stimulated with SDF-1α (100 ng/mL) for 30 seconds, fixed, and stained with rhodamine-labeled phalloidin or Alexa Fluor 550–labeled CTxB (red) and DAPI (blue). The representative images from 3 independent experiments are shown.