FIGURE 3.

Runx1 regulation of Pu.1 corepressor/coactivator exchange is immediate and persistent during Pu.1-driven progressive maturation. Pu.1 was IPed from PUER controls (empty vector pLenti6) and PUER shRunx1 cells at various time points after Pu.1 introduction into the nucleus by OHT. The co-IP was analyzed by Western blot (WB). A, coactivator (Smarca4 and Ncoa5) and corepressor (Sin3a, Chd4, Dnmt1, Lsd1, Eto2, Hdac1, and Hdac2) abundance in the Pu.1 co-IP during progressive macrophage differentiation of PUER controls and PUER shRunx1 cells. Bottom panels show Western analysis of IPed Pu.1 and co-IPed Runx1. Histograms are results of densitometric quantification of co-IPed bands relative to the intensity of IPed Pu.1 bands. B, Pu.1 translocation from the cytoplasm into the nucleus with addition of OHT. C, morphology of PUER control and PUER shRunx1 cells before and 24 h after addition of OHT (macrophage differentiation). Giemsa-stained cytospin preparations of cells at ×200 (microscope model Leica DMR, Leica Microsystems, IL). Images were captured using the attached CRI Nuance NzMSI-FX multispectral imaging system with Nuance software version 2.8 (NuanceCRI). These results were reproduced in three independent experiments.