Macrophage colony stimulating factor

SIRT3 WT and KO mice were kindly provided by Dr. Hyun Seok Kim (Ewha Womans University, Seoul, Korea) and maintained under specific-pathogen-free conditions in Chungnam National University School of Medicine. The mice used in all experiments were 6–8 weeks old and sex-matched. All mice were bred and housed for experiments in accordance with the guidelines of Chungnam National University School of Medicine. Mice experimental protocols were approved by the Institutional Animal Care and Use Committee of Chungnam National University (CNUA-18-0117).
Primary mouse bone marrow-derived macrophages (BMDMs) were isolated and cultured as previously described [16 (link)]. BMDMs were isolated and differentiated after culture for 4–5 days in medium containing 25 ng/ml macrophage colony-stimulating factor (R&D Systems, Minneapolis, MIN, USA). Peritoneal macrophages (PMs) were isolated with 2–3 days after injection with 1 ml of PBS supplemented with 3% thioglycollate (Sigma-Aldrich, St. Louis, MO, USA). Prior to isolation, PMs were collected with pre-chilled PBS supplemented with 10% FBS, in mouse abdominal cavity. BMDMs and PMs were cultured with a medium consisting of DMEM supplemented with 10% FBS, and penicillin-streptomycin-amphotericin B at 37°C, 5% CO₂ atmosphere.

Positively selected CD14+ monocytes were seeded onto tissue culture plates in α-MEM culture media (without ribonucleosides/deoxyribonucleosides) containing 10% FCS, l-glutamine (2 mM), penicillin (50 IU/ml) and streptomycin sulfate (50 μg/ml). Cultures were supplemented with macrophage colony-stimulating factor (R&D Systems, 25 ng/ml), RANKL (gift Dr. J Dunford, 35 ng/ml) and experimental treatments either OPG (Peprotech, 500 ng/ml), LDN193189 (Sigma, 100 nM), human BMPR1a-FC (gift Dr. E. Martinez Hackert 10 µg/ml), or human ACVR2a-FC (gift Dr. E. Martinez Hackert 10 µg/ml), every 3–4 days, with mature osteoclasts being formed by day 9. Cells were fixed in 4% formalin and tartrate-resistant acid phosphatase (TRAP) was visualised using naphthol AS-BI phosphate as a substrate, with reaction of the product with Fast Violet B salt. TRAP-positive multi-nucleated cells containing ≥3 nuclei were considered osteoclasts.

Lung cancer cell lines A549 and H460 and murine macrophage cell line RAW264.7 were purchased from the Korean Cell Line Bank (Seoul, Korea). A549, H460, and RAW264.7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Welgene, Gyeongsan, Korea) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific), 100 U/mL penicillin, and 100 U/mL streptomycin at 37 °C with 5% CO₂. A549 and H460 cells were used in passage 10–13, and RAW264.7 cells were used in passage 60. BMDMs were isolated from the femur and tibia bones of 6–8-week-old C57BL/6 mice and differentiated after four days in a medium containing macrophage colony-stimulating factor (25 ng/mL; R&D Systems, Minneapolis, MN, USA) at 37 °C with 5% CO₂.