c-Myc is a downstream target of Rbm15 in the regulation of HSC and megakaryocyte development. (A-B) c-Myc expression levels are regulated by Rbm15 and RBM15-MKL1 in HSCs. (A) Expression of selected genes in Rbm15-KO HSCs. RNA was isolated from FACS-sorted LSK-HSCs from both Rbm15-deleted and WT littermate control mice, reverse transcribed, and analyzed by TaqMan real-time PCR. Normalized expression of each gene in Rbm15-deleted cells relative to that in WT control LSK cells was calculated. Results represent the mean ± SD of 3 independent experiments. (B) c-Myc levels in HSCs with ectopic RBM15 and RBM15-MKL1 expression. LSK-HSCs were FACS sorted and transduced with MSCV-IRES-GFP (MIG) vector control, MSCV-IRES-GFP-RBM15 (MIG-RBM15), or MSCV-IRES-GFP-RBM15-MKL1 (MIG-RBM15-MKL1) retrovirus supernatant. After a 2-day transduction, GFP-positive cells were sorted for real-time PCR analysis (mean ± SD, n = 2). (C) c-Myc overexpression rescues the megakaryocyte increase in ex vivo cultures. Lin⁻ cells were isolated by magnetic-activated cell sorting from Rbm15-KO and -WT littermate control animals and transduced with either MSCV-IRES-GFP (MIG) or MSCV-IRES-GFP-c-Myc (c-Myc) retroviruses before culturing ex vivo using the same conditions as described in Figure 5. GFP⁺ cells were gated and megakaryocytes were analyzed using the specific cell surface marker CD41. The results shown are representative of 3 independent experiments.