Prostaglandin e2

Natural Medicine Research Center of Jilin University provided protopanaxatriol saponins (PTS; batch number 210115). Dextran sodium sulfate (DSS; MW: 36,000–50,000 Da) was acquired from MP Biomedicals company (Santa Ana, CA, USA). The Chang Yan Ning (CYN; a positive control) table was acquired from Jiangxi CONBA Chinese Medicine Co, Ltd. (Shangrao, China). Ginsenosides Re and Rg1 were acquired from Natural Medicine Research Center of Jilin University. Riboflavin, prostaglandin E2, PC (16:0/16:0), and prostaglandin D2 with purity ≥ 95% were acquired from Merck (Darmstadt, Germany). Myeloperoxidase (MPO), interleukin-1β (1L-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) enzyme-linked immunosorbent assay kits were purchased from Hangzhou Lianke Biotechnology Co., Ltd. (Hangzhou, China). A nitric oxide detection kit and 4% paraformaldehyde solution were acquired from Biyuntian Co., Ltd. (Shanghai, China).
Methanol and acetonitrile of UPLC grade were acquired from Thermofisher Scientific (Shanghai, China). We bought deionized water from A.S. Watson Bunch Ltd. (Hong Kong, China). All other chemical solvents were of analytical grade.

Bone marrow cells were cultured in α-MEM containing 10% FBS, 100 unit/ml penicillin and 100 μg/ml streptomycin for 24 h to obtain BMMs. The BMMs were then cultured on coverslips in α-MEM containing 20 ng/ml M-CSF (R&D Systems, Inc., MN, USA) for 2 days and in the same medium containing 20 ng/ml M-CSF, and 3.3 ng/ml RANKL (R&D Systems, Inc., MN, USA) for an additional 6 days. TRAP-positive spreading osteoclasts containing more than 5 nuclei were quantified using OsteoMeasure software.
For co-culture studies, osteoblasts derived from the long bones of FcγRIIB^−/− males and their WT controls were cultured in α-MEM containing 10% FBS, 100 unit/ml penicillin and 100 μg/ml streptomycin for 24 h. BMMs from either FcγRIIB^−/− mice or WT controls were added and cultured in α-MEM containing 10⁻⁶ M prostaglandin E₂ (Merck Millipore, Murlington, MA, USA) and 10⁻⁸ M 1,25-dihydroxyvitamin D₃ (Merck Millipore, Murlington, MA, USA) for 4 days. TRAP-positive spreading osteoclasts containing more than 5 nuclei were counted using OsteoMeasure software.

Lentiviruses were produced by transfection in 293T with MISSION shRNA vectors (Sigma-Aldrich). CML CD34⁺ cells (4 × 10⁶ cells/ml) were infected by spinoculation (800 g, 90 min, 30 °C). Two adjuvants, poloxamer 407 (100 μg/mL, Sigma-Aldrich) and prostaglandin E2 (10 μM, Merck), were added to the culture medium [50 (link)]. Cells were harvested 72 h later, and the knockdown effect was examined by western blot analysis.