Coexpression of SOCS1 and FLT3-ITD shields colony growth from inhibition by interferon α and γ. Primary murine bone marrow was retrovirally transduced with empty vector control, SOCS1, FLT3-ITD, or SOCS1-T2A-FLT3-ITD and was sorted for GFP coexpression. (A) After RNA extraction and reverse transcription, quantitative PCR was performed to determine endogenous and exogenous levels of SOCS1, normalized to RNA polymerase IIa. (B) Cell lysates were resolved by SDS-PAGE and immunoblotted for (total) FLT3, SOCS1, GFP, and β-actin (loading control). Exogenous SOCS1 (right lane) slightly larger in size because of few additional amino acids resulted from T2A cleavage. (C) Sorted GFP–positive bone marrow cells were washed and plated at a density of 2000 cells/mL in triplicates in methylcellulose medium containing either proproliferative growth factors (IL-3, SCF, and IL-6) or IFN-α (300 U/mL) or IFN-γ (100 ng/mL). Colony numbers were counted after 12 days, and mean colony number from triplicates from 2 independent experiments were plotted, with SD as error bars. (D) Sorted, FLT3-ITD–expressing bone marrow cells were transduced with either SOCS1-specific shRNAs^1–5  or a nonspecific shRNA and selected in the presence of puromycin. Puromycin-resistant clones were analyzed for SOCS1 protein expression, parental bone marrow expressing FLT3-ITD (not transduced with shRNA vectors) was used as a control, and β-actin was detected as a loading control. (E) Sorted and puromycin-resistant (FLT3-ITD and shRNA expressing clones 3 and 4) bone marrow cells or FLT3-ITD–expressing control cells were washed and plated at a density of 2000 cells/mL in triplicates in methylcellulose medium containing either proproliferative growth factors (IL-3, SCF, and IL-6) or IFN-α (300 U/mL) or IFN-γ (100 ng/ mL). Colony numbers were counted after 12 days, and mean colony number from triplicates are plotted, with SD as error bars (*P < .05).