Effects of RhoH overexpression on F-actin polymerization, cortical F-actin localization, and migration of hematopoietic progenitor cells. The transduced and EGFP⁺, c-Kit⁺–sorted cells were used in the following assays. (A) F-actin subcellular localization. Cells were stimulated with SDF-1α (0 or 100 ng/mL) for 30 seconds before stained with rhodamine-labeled phalloidin (red) and 4′,6-diamidino-2-phenylindole (DAPI; blue). Fluorescence images (original magnification, × 400) were acquired on a Leica microscope with a deconvolution system. Images are representatives of more than 100 cells examined for each construct. Cortical F-actin localization is shown by arrows. (B) Quantitative analysis of F-actin polymerization by flow cytometry. Cells were stimulated with 100 ng/mL SDF-1α and stained with phalloidin–Alexa Fluor 633 for F-actin content. Data represent the percentage of F-actin content (MFI) over unstimulated vector-transduced cells. (C) Migration in a transwell chamber assay in response to SDF-1α (0-1000 ng/mL). Data represent the percentage of the migrated cells as the mean ± SD; n = 3. *P < .01, RhoH-versus vector-transduced cells. These results shown in each panel are representatives of 3 experiments.