BV2 mouse microglial cell line was kindly provided by Marc Diamond (UT Southwestern Medical Center). BV2 cells were cultured in DMEM (Gibco 11965) supplemented with 5% FBS (Sigma F4135) and 100 U/ml penicillin-streptomycin (Gibco 15140). Routine testing of cell lines using MycoAlert PLUS mycoplasma detection kit (Lonza) showed that BV2 cells were negative for mycoplasma contamination. pcDNA3-FLAG-PU.1 was a gift from Christopher Vakoc79 (Addgene plasmid 66974). pGFP-V-RS with either non-targeting shRNA or PU.1-targeting shRNAs was purchased from OriGene Technologies (TG502008). The pHrodo red zymosan conjugate bioparticles from Thermo Fisher (P35364) were used to assess phagocytic activity. For transient transfections, 200,000 cells were seeded in a 24-well plate. On the next day, cells were washed with PBS (Gibco 14190) and medium was changed to 400 μl DMEM supplemented with 2% FBS without antibiotic. Transfection mixes of 0.5 μg pcDNA3 or 0.5 μg pcDNA3-FLAG-PU.1 with 0.5 μg pCMV-GFP for overexpression of mouse PU.1 and 1μg pGFP-V-RS-shSCR, -shA, -shB and -shD for knock-down of mouse PU.1 were prepared with 2 μl of Lipofectamine 2000, incubated for 20 min at room temperature and added
