Efficient immortalization of myeloid progenitors by Setbp1 expression. (A) Schematic diagram of the immortalization procedure. (B) Representative cytospin preparation and Wright-Giemsa staining of cells infected with Setbp1 (top panel) or empty retrovirus (bottom panel) at 1 month after infection. Original magnification ×400. Images were obtained using a Nikon Eclipse E800 microscope and a Qimaging Micropublisher 5.0 digital camera. (C) Cytospin preparation of Setbp1-immortalized cells (BM70 and P3 population) before and after treatment with G-CSF for 2 days. BM70 cells are immortalized by insertional activation of endogenous Setbp1.^12,13  Original magnification ×400. (D) Southern blotting analysis of viral integrations present in 6 Setbp1-immortalized myeloid progenitor populations (P1-P6) using a GFP-specific probe. Seven ug of genomic DNA from each population was digested with EcoRI, resulting the generation of a single GFP-containing DNA fragment from each provirus. Each band represents an independent integration. (E) Real-time RT-PCR analysis of total RNA isolated from purified LSK, CMP, and GMP populations of C57BL/6 mice using Setbp1-specific primers. Relative expression levels were calculated by normalizing to β-actin mRNA levels in the same sample and also in LSK cells. The mean and SD of each relative expression level is shown.