SHP2 knockdown suppresses 32Dcl3 cell granulopoiesis. (A) 32Dcl3 cells were removed from IL-3 for 3 hours and than exposed to 20 ng/mL G-CSF for 0, 5, or 10 minutes. Total cellular proteins were collected and then subjected to Western blotting for phosphorylation of SHP2 on Y580 (P-SHP2), total SHP2, and β-actin. (B) 32Dcl3 cells were transduced with a panel of lentiviral vectors expressing SHP2 shRNAs(74-78) or with the empty vector (Puro), and pooled transductants were selected. Total cellular proteins were then subjected to Western blotting for SHP2 or β-actin. (C) Similar Western blot analysis was conducted using total cellular protein samples prepared from parental 32Dcl3 cells (32D), vector-transduced cells, subclones of the 32Dcl3(shRNA-77) pool (10,12,13,15,16), and the shRNA-77 cellular pool. (D) Growth curves show daily viable cell counts for the cells transduced with empty vector and for the indicated shRNA-77 subclones. (E) The vector-transduced cells or the 5 32Dcl3(shRNA-77) subclones were transferred to G-CSF and total cellular RNAs were prepared after 24 hours. These were converted to first-strand cDNA and the relative expression of MPO, PR3, and LF, normalized to ribosomal protein S16 RNA expression, were analyzed by real-time PCR analysis. Shown are means and SE from 3 independent RNA preparations. * indicates P < .05 for each RNA level relative to expression in the vector-transduced cells.