Murine rankl

Marrow cells were flushed from the right femur and tibia, washed, resuspended in complete α‐MEM (containing 10% FBS, 2 mm l‐glutamine, 50 IU/ ml penicillin, and 50 mg/ml streptomycin sulphate), and seeded into 24‐well plates at 5 × 10⁵ cells per well. After 2 h incubation, non‐adherent bone marrow cells were reseeded onto dentine discs or plastic dishes and treated with M‐CSF (25 ng/ml) and murine RANKL (50 ng/ml; Peprotech, London, UK) every 3–4 days for 9 days to induce osteoclast formation.
Adherent marrow cells were supplemented to encourage osteogenic differentiation (50 μg/ml ascorbic acid phosphate, β‐glycerophosphate 2 mM, dexamethasone 10 nm) or adipogenic differentiation (100 μm oleic acid). Media were changed every 3–4 days for 28 days. Osteoblast cultures were fixed on day 14 for alkaline phosphatase staining, using naphthol AS‐MX phosphate as a substrate and reaction of the product with Fast Violet B salt, and on day 28 for assessment of mineralization by staining with Alizarin Red S. Plates were scanned and the area and intensity of staining were quantified using ImageJ. Adipocyte cultures were also fixed on day 28 for assessment of triglyceride levels by staining with Oil‐Red‐O, which was extracted with isopropanol, and absorbance was measured at 510 nm.

Mammary glands were mechanically dissociated, finely minced and digested for 1 h at 37 °C in DMEM/F-12 medium (Invitrogen) containing 2 mg/ml collagenase A (Roche Diagnostics) and 100 U/ml hyaluronidase (Sigma-Aldrich) on a shaking platform at 140 rpm. Organoids were washed in PBS and separated from single cells through four differential centrifugations (pulse to 1500 rpm in 10 ml DMEM/F12), as previously described^{53 (link)}. The pellet was then re-suspended in Phenol-free Growth Factor Reduced 100% Matrigel (BD Biosciences), and plated in eight-well coverslip bottom chambers (Fisher Scientific) and kept for 20 min at 37 °C before addition of DMEM-F12 culture media containing Transferrin/Sodium Selenite (Sigma-Aldrich), 5 µg/ml insulin (Sigma-Aldrich) and 1% Penicillin/Streptomycin (Life Technologies). After 24 h, 5 nM 4-OHT (Sigma-Aldrich) was added for 6 h. For testing hormone responses, the medium was supplemented with 200 ng/ml murine Rankl (PeproTech) or 25 ng/ml of progesterone (Sigma-Aldrich) and 50 nM 17β-estradiol (Sigma-Aldrich). Cells were cultured for 7 days.

For osteoclastic differentiation, cells were cultured in the presence of 25 ng/ml murine M-CSF and 50 ng/ml murine RANKL (for RAW264.7, from PeproTech, USA), human M-CSF and human RANKL (for PBMCs, from Peprotech, USA), and RPR. In some experiments, cells were pre-incubated for 40 mins with pharmacologic inhibitor p38/MAPK (SB203580; 10 ng/ml), ERK1/2 (PD98059; 20 μM), and JNK (SP600125; 10 ng/ml) (all from Calbiochem, La Jolla, CA) before RPR was added. In other experiments, cells were pre-incubated for 30 mins with an NFκB inhibitor (NF-κB SN50, cell-permeable inhibitor peptides; Calbiochem, San Diego, CA); caspase-3 specific inhibitor, Z-DEVD-FMK (R&D Systems, Inc., USA); caspase-9 specific inhibitor, Z-LEHD-FMK (BD Biosciences, San Diego, CA); or the general caspase inhibitor, Z-VAD-fmk (Bachem, Bubendorf, Sweden) at a concentration of 20 μM before RPR treatment.

Animal use was approved by the Ethics Committee at Tianjin Medical University. We used 123 C57BL/6 female mice in total (~16 wks, Animal Center of Academy of Military Medical Sciences, China). Under pathogen-free conditions, 4–5 mice were housed per cage, and they were maintained at 25 °C on a 12-h light/dark cycle, with standard rodent chow and water ad libitum.
We purchased a murine RANKL (receptor activator of nuclear factor kappa-B ligand) and murine M-CSF (macrophage-colony stimulating factor) from PeproTech, and Dulbecco’s Modified Eagle’s Medium (DMEM), Minimum Essential Medium Alpha (MEM-α), fetal bovine serum (FBS), penicillin, streptomycin, and trypsin from Invitrogen (Carlsbad, CA, USA). Other agents were obtained from Sigma (St. Louis, MO, USA) unless otherwise stated.