MITF is essential for BMP4-mediated increase in ITGA4 expression, and HSPC homing. (A) Schematic representation of Mitf knockdown experiments. Lin− cells were transduced with a lentiviral vector containing doxycycline inducible anti–Mitf-shRNA or nonsilencing construct (NSC). Expression of the shRNA was induced with doxycycline and the transduced cells were selected using hygromycin. Expression of the anti–Mitf-shRNA or NSC was detected by RFP expression. Mitf-KD and NSC Lin− progeny were then cultured with or without BMP4 and evaluated by qRT-PCR, Western blotting, FACS and in vivo homing assays. (B) qRT-PCR for Mitf and Itga4 expression (n = 3; P < .02). (C) Western blotting using antibodies against MITF and β-actin. Representative example of 3 independent experiments. (D) FACS analysis with antibodies against ITGA4. Representative example of 3 experiments. (E) KLS cell progeny (0.1 × 106) from the different cultures were transplanted intravenously in lethally irradiated mice, and the BM harvested after 16 hours. Total BM cells (0.1 × 106) were assayed in methylcellulose colony forming assay, Percentage homing was determined by comparing total CFU-Cs homed with CFU-Cs injected (n = 3, P < .02).
