Role for STAT1 in IFN-α–mediated pDC development. (A) STAT activation and expression in IFN-α–, GM-CSF-, or IFN-α + GM-CSF-treated (30 minutes) DC progenitors (lin⁻ Flt3⁺ cells), determined by immunoblotting, as indicated. (B) Proportion of pDCs (top left quadrant) and cDCs (bottom right quadrant) in total BM cultures from Stat1^−/− or wild-type mice in GM-CSF or GM-CSF + IFN-α for 4 days. Results were gated on the CD11c⁺ population (not shown); B220 and CD11b analysis is shown. n = 5. (C-D) Proportion (C) and absolute numbers (D) of pDCs and cDCs in BM and spleen of Stat1^−/− or wild-type mice treated by HGT, as indicated. Results were analyzed as indicated in panel B. n = 5. Error bars represent SEM. (E) Gene expression in D2SC/1 cells stimulated with GM-CSF, IFN-α, or both for 2 hours or left unstimulated, as indicated. Error bars represent SEM. (F) EMSAs with nuclear extracts from D2SC/1 cells stimulated with or without IFN-α for 30 minutes. Some samples contained STAT1 competitor antibody or competitor oligonucleotides, as indicated. d indicates Irf8 STAT site; m, mutated Irf8 STAT site; and s, STAT1-consensus oligonucleotide. (G) Luciferase assays in IFN-α–stimulated (IFN) or untreated (NT) D2SC/1 cells transfected with empty vector (pGL4.12) or an Irf8 reporter (pGL4.12/IRF8) with an intact (WT) or mutated (MU) STAT element, plus pMNC/mSTAT1, pMNC/mSTAT2, and phRL-TK plasmids. Error bars represent SEM. (H) ChIPs from D2SC/1 cells with or without IFN-α stimulation (1 hour) with STAT1 antibodies or IgG controls, as indicated. PCR reactions were performed with total cell lysates (input) or immunoprecipitated samples, as shown. (A-H) Results represent 3 independent experiments.