In Vitro Osteoclastogenesis Assay Protocol

In vitro osteoclastogenesis assays were performed as described previously^{18 (link),112 (link)} with modifications. Briefly, mouse bone marrow cells were harvested from 4-week-old male C57BL/6J mice by flushing marrow cavity from both tibia and femur, filtered through 40 µm cell strainer, and cultured with alpha minimum essential medium (α-MEM) containing 15% fetal bovine serum (FBS), 100 U/mL streptomycin sulfate (Sigma-Aldrich) and 100 U/mL penicillin (Sigma-Aldrich) in 10-cm culture dishes at 37 °C in 5% CO₂ humidified incubator for at least 24 h following established protocols. The adherent cells were discarded while the floating cells were cultured with macrophage colony-stimulating factor (M-CSF; R&D Systems) at 30 ng/mL for 48 h. To collect pure monocytes/macrophages, the cells are digested with Versene for ~4 min in 37 °C until most macrophages are de-associated. Then the cells are seeded in 24-well plate and cultured with 30 ng/mL M-CSF and 100 ng/mL RANKL (Abcam, ab129136) for 5 days to induce the formation of mature osteoclasts. For treatments, 10⁻⁶ M MPS with/without 10⁻⁶ M RU486 is added to culture medium at designated timepoints and cultured for 24 h. TRAP activity is measured using a commercial kit (Sigma-Aldrich). Total RNA was extracted, and the mRNA level of ANG was measured by qRT-PCR.