Regulation of myeloproliferation and M2 macrophage programming in mice by Lyn/Hck, SHIP, and Stat5
J. Clin. Invest. Wenbin Xiao, et al. 118:924 doi:10.1172/JCI34013 [Go to this article.]

Figure 4
Stat5 activation is required for the increased proliferation and macrophage differentiation of KSL cells from Lyn/Hck-deficient mice. (A and B) Stat5 in dko KSL cells was constitutively activated (–), and its activity was further increased upon stimulation with a cytokine cocktail of IL-3, SCF, and Flt3 ligand (+), as shown by flow cytometric analysis (A) and confocal microscopy (B) using anti–p-Stat5 (Tyr694) antibody. Nuclei were stained with DAPI (blue). Original magnification, ×400. One hundred cells were scored for either the absence (type I) or presence (type II) of anti–p-Stat5 staining and those numbers are shown to the right of the panel. (C–E) Stat5 in dko lung macrophages was constitutively phosphorylated, as shown by flow cytometric analysis (C), confocal microscopy (D), and immunohistochemistry (E). Original magnification, ×400 (D and E). (F) Real-time PCR was performed for mRNA expression of Stat5 target genes, cytokine-inducible SH2-containing protein (CIS), pim-1, and oncostatin M (onc M). Expression was normalized against L32 RNA and set at 1 in WT cells. *P < 0.05; **P < 0.01. (G) Dko and WT KSL cells were transduced with a bicistronic retroviral vector encoding DN Stat5, DN Stat3, or empty vector, together with GFP, and cultured in medium containing SCF, Flt3 ligand, and IL-11. Ratios of cell numbers normalized against those of empty vector-transduced cells are plotted. For example, “WT (DNStat5/vec)” indicates the ratio of the numbers of WT cells transduced by DN Stat5 to those of WT cells transduced by empty vector. ^#P < 0.05 versus WT (DNStat5/vec). (H) Transduced KSL cells (150 cells seeded) were cultured in methylcellulose medium containing IL-3, IL-6, SCF, and erythropoietin. BFU, BFU-E; G, G-CFU; M, M-CFU; GM, GM-CFU; Mix, Mix-CFU.