Effects of enforced expression of PU.1 on cell-surface expression of CD80 and CD86. (A) Schematic drawing of plasmids pMX-IG (IG-mock) and pMX-IG-PU.1 (IG-PU.1) used in Figures 5C-D and 7D). (B) Schematic drawing of plasmids pMX-puro (mock), pMX-puro-PU.1 (puro-PU.1), and pMX-puro-Ets (puro-Ets) used in Figure 5E and F. Lin⁻ cells were transfected with the pMX-IRES/GFP series and transfectants were monitored as GFP-positive cells. Cells were incubated with infectious viruses for 3 days in the presence of IL3, IL6, SCF, TPO, and EPO with (C) or without (D) GM-CSF, G-CSF, M-CSF, and Flt3L. Three days after infection, cells were stimulated with or without 1 μg/mL of LPS and/or 100 ng/mL of IFNγ for 24 hours, followed by staining with PE-labeled mAbs, and transfectants were monitored as GFP-positive cells (C-D). Lin⁻ cells transfected with the pMX-puro series were incubated with the cytokine mixture as in panel C (for E) or panel D (for F). To concentrate transfectants, cells were maintained for 10 days in the presence of puromycin (2 μg/mL). Solid line histogram represents cells with each antibody. Dotted line histogram represents negative control with 2.4G2 alone. Representative results are shown. (F) Western blotting to compare endogenous and exogenous PU.1 protein levels in retrovirus transfectants. Green, anti–Flag antibody; red, anti–PU.1 antibody.