Effects of inhibition of endogenous RhoH expression by RNAi on hematopoietic progenitor cell proliferation, survival, and migration. (A) MIEG3-based retroviral vector expressing EGFP and siRNAs specific to RhoH (the siRhoH or the control siRhoH) driven by a U6 gene promoter. Wild-type LDBM cells were transduced with MIEG3/U6-siRhoH retroviral vectors. The EGFP⁺, c-Kit⁺ cells were sorted and used in the following assays. (B) The levels of RhoH and Rac2 mRNA examined by a quantitative real-time RT-PCR assay. Endogenous c-Abl expression was used as an internal control for quantification. (C) The level of RhoH protein examined by immunoblot analysis using a polyclonal anti-RhoH antibody. The level of Rac proteins was used as a control. (D) Myeloid progenitor colony-forming unit assay. Cells were plated in methylcellulose in the presence of SCF, G-CSF, and MGDF. (E) Cell expansion in liquid culture in response to SCF. (F) SCF-induced proliferation measured by [³H]-thymidine incorporation. (G) Apoptosis in the presence of SCF analyzed by Annexin V-APC and 7-AAD staining and flow cytometry. (H) Migration in a transwell chamber assay in response to SDF-1α (0-1000 ng/mL). In panels B and D-H, data represent the mean ± SD; n = 3. *P < .01 or P < .05, RhoH-versus vector- or control siRhoH-transduced cells. Results are representatives of a minimum of 3 experiments.