IL-3 induces BM-derived CD45⁺ cell proliferation via STAT5 activation. (A,B) The percentage of cells in the S phase was evaluated by FACS analysis on human (A) or mouse (B) BM-derived CD45⁺ cells cultured as indicated. (C) FACS analysis was performed on IL-3–cultured BM-derived CD45⁺ cells. Black lines, preimmune IgG (negative control); gray lines, Tie2, KDR, and CD33 markers. (D) Representative photomicrographs of 8 days IL-3–cultured cells. Magnification, 10× (panel i) and 40× (panel ii). (E) Representative sample of IL-3–cultured BM-derived CD45⁺ cells labeled with Dil-acLDL. Phase-contrast (panel i) and fluorescence (panel ii). Magnification, 10×. (F) Cell extracts from BM-derived CD45⁺ cells, cultured with or without IL-3 as indicated, were subjected to SDS-PAGE. The filter was IB with anti-p-STAT5 and anti-STAT5 antibodies. (G) IL-3–cultured BM-derived CD45⁺ cells transfected with STAT5A or STAT5B siRNA, alone or in combination, or with the scrambled sequence (scramble) were lysed. Cell extracts were analyzed by WB with anti–STAT5, anti–cyclin D1, and anti–β-actin antibodies (left panel). IL-3–treated ECs were used as positive control (+). In selected experiments, IL-3–cultured CD45⁺ cells, transfected with STAT5A and/or STAT5B DN constructs or with the empty vector (pcNEO), were assayed for the expression of p-STAT5, cyclin D1, and β-actin (right panel). (H) The percentage of cells in the S phase was evaluated by FACS analysis on BM-derived CD45⁺ cells transfected with STAT5A and/or STAT5B DN constructs or with the pcNEO vector (*P < .05, experimental groups vs pcNEO). Three different experiments were performed with comparable results.