In Runx1-deficient (shRunx1) cells, Pu.1 (OHT) repressed stem cell genes but myeloid differentiation gene activation was impaired. (A) The pattern of mRNA expression in PUER shRunx1 compared with PUER empty vector (control) cells treated with OHT. Gene expression after OHT addition was measured by quantitative RT-PCR. Stem cell genes are Bmi-1, Hoxb4, and c-Kit. Differentiation genes are F4/80, Mcsfr, and Gmcsfr. Data are mean ± SD. shRunx1 versus control for each time point: *P < .05, **P < .01 (Student t test). Experiments were performed in triplicate. (B) The pattern of c-Kit and F4/80 protein expression. Measured by flow cytometry 48 hours after OHT. MFI indicates mean fluorescence intensity of all cells. (C) OHT effect on cell proliferation of shRunx1 and control cells. Cell counts were measured daily with an automatic cell counter. Experiments performed in triplicate. Error bars represent SE. (D) OHT effect on cell morphology of shRunx1 and control cells. Cell morphology was evaluated on Giemsa-stained cytospin slides 48 hours after OHT. Microscope: Leica DMR, 63×/1.32 oil PH3 type N immersion oil. Image capture: CRI Nuance NzMSI-FX with Nuance 2.8 software. (E) Decreased histone acetylation at Gmcsfr and Mcsfr proximal promoters in shRunx1 compared with control cells. The Gcsfr promoter, which is not known to be regulated by Runx1/PU.1, was analyzed as control. ChIP performed with anti-H3Ac. Coimmunoprecipitation of promoter regions was analyzed by quantitative RT-PCR. Data are mean ± SD. shRunx1 vs control for each time point after OHT: *P < .05, **P < .01 (Student t test). Experiments performed in triplicate.