Exogenous C/EBPα rescues granulopoiesis and exogenous RUNX1 rescues C/EBPα expression after SHP2 knockdown. (A) 32Dcl3(shRNA-77)-10 cells were transduced with pBabeNeo (Neo) or pBabeNeo-C/EBPα-ER (αER). Total cellular proteins prepared from these cells or from 32Dcl3-Puro cells were subjected to Western blotting for C/EBPα or β-actin. The positions of C/EBPα or C/EBPα-ER are shown. (B) Total cellular RNAs prepared from 32Dcl3(shRNA-77)-10+αER cells 0 or 1 day (D1) or 4 days (D4) after addition of estradiol (E2) were analyzed for relative expression of the mRNAs encoding MPO, PR3 or LF. The mean relative expression of each mRNA after estradiol addition rescue was set to 1.0. Results shown are mean and SE of 3 repetitions. * indicates P < .05 for each RNA comparing expression in the presence versus absence of estradiol. (C) 32Dcl3(shRNA-77)-10+αER cells were cultured with estradiol for 0 or 4 days, cytospun, and subjected to Wright-Giemsa staining. (D) 32Dcl3(shRNA-77)-10 cells were transduced with or pBabeNeo-RUNX1-ER (Rx1ER). Total cellular proteins prepared from these cells, from 32Dcl3-Puro cells, or from 32D(shRNA-77)-10+Neo cells were subjected to Western blotting for RUNX1 or β-actin. (E) Total cellular proteins prepared from these same 3 cell lines cultured ± 4HT for 24 hours were subjected to Western blotting for C/EBPα or β-actin.