Human cd14 monocytes

THP-1 monocytes were purchased from ATCC (TIB-202) and grown in “THP-1 media” consisting of RPMI 1640 (ATCC modification; Thermo, A1049101) supplemented with 10% defined FBS (HyClone, SH30070.03), 1x penicillin/streptomycin (Thermo, 15140-122), and 0.05 mM beta mercaptoethanol (Sigma, M3148). Bone marrow-derived macrophages (BMDM) were cultured from bone marrow of 6 to 10-week-old C57BL/6 mice. Bone marrow cell suspensions from femurs and tibias of C57BL/6 mice were grown for 7 days in non-treated sterile 10 cm plates in “BMDM media” consisting of DMEM/F-12 (Thermo, 10565-018) supplemented with 10% heat-inactivated defined FBS, 1× penicillin/streptomycin, 1× non-essential amino acids (Thermo, 11140-050), and 20 ng mL⁻¹ mouse M-CSF (BioLegend, 576406). Mouse peritoneal and alveolar macrophages were isolated from 6- to 10-week-old C57BL/6 mice⁵⁰ . Human CD14⁺ monocytes were purchased from Lonza and grown in RPMI (Sigma, R8758) supplemented with 10% heat-inactivated defined FBS, 1x penicillin/streptomycin and 50 ng mL⁻¹ human recombinant M-CSF (Biolegend, 574804) for 7 days. All cells were incubated in a 5% CO₂ incubator at 37 °C unless otherwise noted. All animal experiments were performed in accordance with an approved protocol from the Institutional Animal Care and Use Committee (IACUC) of RIKEN Yokohama Branch.

Primary human umbilical vein endothelial cells (HUVECs) were obtained from Cell Applications (San Diego, CA). and cultured in medium 200 (Thermo Fisher Scientific, Waltham, MA) supplemented with Low Serum Growth Supplement (LSGS, Thermo Fisher Scientific) and gentamicin/amphotericin B. Human acute monocytic leukemia cells (THP-1) were obtained from ATCC (Manassas, VA). THP-1 cells were maintained in RPMI 1640 (ATCC) medium supplemented with 10% fetal bovine serum (Thermo Fisher Scientific), 1% penicillin/streptomycin (Thermo Fisher Scientific) and 0.05 mM 2-mercaptoethanol (Sigma Aldrich). Human CD14 + monocytes and primary human coronary artery endothelial cells (HCAECs) were obtained from Lonza (Walkersville, MA). HCAECs were cultured in microvascular endothelial cell growth media (EGM-2 MV, Lonza) according to provider’s instructions. Interferon-γ (IFN-γ) and Tumor Necrosis Factor-α (TNF-α) were purchased from R&D Systems (Minneapolis, MN). For knockdown of QKI, monocytes were cultured as above and treated with either a non-targeting control oligonucleotide or one three different QKI targeting FlexiTube siRNAs (Qiagen, Germantown, MD) for 18 h prior to preparation of total RNA.