SHP2 knockdown suppresses granulopoiesis and favors monopoiesis in murine marrow cells. (A) Diagram of experimental protocol. Marrow cells were lineage-depleted, placed in TPO, FL, and SCF, transduced with pLKO.1 (Puro) or pLKO.1-shRNA-77, and then exposed to puromycin for 3 days. Viable cells were then transferred to IL-3, IL-6, and SCF on day 0 (D0), in liquid culture for FACS or RNA analyses or in methylcellulose for assay of myeloid colony-forming units (CFU). (B) Growth curves show viable cell counts over a 5 day period in TPO, FL, and SCF and daily after transfer to IL-3, IL-6, and SCF. (C) Western blot analysis of SHP2, C/EBPα, and β-actin expression on Day 0 (left). Relative intensity of SHP2 or C/EBPα bands is shown, normalized to β-actin. Expression of CEBPA RNA on day 0 (right), normalized to ribosomal protein S16 mRNA expression (mean and SE from 3 determinations). (D) Representative FACS analysis for Mac-1 and Gr-1 expression on Day 2 and the increase in the Mac-1⁺Gr-1⁻ monocyte (M)/Mac-1⁺Gr-1⁺ granulocyte (G) ratio (mean and SE, with the M/G ratio set to 1 in each of 3 experiments). (E) Total cellular RNAs prepared on day 2 were analyzed for expression of the indicated mRNAs, normalized to S16 mRNA expression. The ratio of expression of each mRNA in cells transduced with SHP2 shRNA-77 relative to expression in cells transduced with empty vector is shown (mean and SE from 3 determinations). * indicates P < .05 relative to a ratio of 1.0. (F) Cells on day 0 or day 3 after transfer to IL-3, IL-6, or SCF were cytospun and subjected to Wright-Giemsa staining. (G) The proportion of CFU-G relative to CFU-G + CFU-M after culture of cells transduced with empty vector or shRNA-77 is shown (mean and SE from 3 determinations). Average number of CFU-G+CFU-M was 113/10 000 cells in the vector group and 30/10 000 cells in the shRNA-77 group. P < .01 for shRNA-77–expressing cells relative to vector transduced cells.