PU.1 regulates miR-146a in vivo. (A) MiR-146a expression in WT hematopoietic precursors and mature myeloid cells. miR-146a qRT-PCR was performed from FACS-purified HSCs (LSKs), common myeloid progenitors (CMPs; Lin⁻Sca-1−c-Kit⁺CD34⁺FcγRII/III^low), splenic granulocytes (Gra; Gr1⁺CD11b⁺), and splenic macrophages (Mac; Gr1⁻CD11b⁺). Data were compared with U6 RNA ± SD miR-146a and PU.1 mRNA expression in the indicated FACS-purified hematopoietic populations from WT and PU.1 KD animals. (B) MiR-146a was analyzed compared with U6 RNA, and PU.1 expression was determined relative to β-actin. **P < .001 by Student t test. (C) A 375-bp fragment containing the PU.1-binding site at 10 kb upstream of the genomic miR-146 locus was inserted in the pXP2 luciferase plasmid pXP2-(-)10kb. Mutation of the PU.1 motif (ctgaggaagt to ctgaccaagt) in the pXP2-(-)10kb construct, as indicated by the crossed-out PU.1 symbol in the pictogram, led to a marked reduction in reporter activity in transiently transfected myeloid RAW264.7 cells. Relative expression was determined 48 hours after transfection compared with pXP2 empty basal fluorescence activity (n = 3). **P < .001 by Student t test. (D) RAW264.7 cells carrying a stable PU.1 KD shRNA showed significantly lower miR146 promoter activity than plain RAW264.7 cells when transiently transfected with the WT pXP2-(-)10kb construct. Reporter activity of plain RAW264.7 cells was set to 100% (n = 3). **P < .001 by Student t test. (E) PU.1 expression in untreated and stably PU.1-shRNA–transfected RAW264.7 cells in Western blot normalized to levels of tubulin. Error bars indicate SD.