Recombinant mouse il 1β

BMDMs were harvested and cultured from the indicated mouse strains. Briefly, bone marrow was flushed from the bones of the hind legs and cultured for 6–10 days in non-tissue-treated dishes in DMEM with 10% FCS plus conditioned media from L929 cells at a 1:5 ratio53 (link). BMDM CM was collected after 72 h of 100 ng ml⁻¹ IL-1β treatment. Primary mouse pre-adipocytes were isolated from the mammary fat pad of 4-day-old mice and cultured in DMEM supplemented with 10% FCS, non-essential amino acids (Thermofisher, Waltham, MA), Glutamax (Thermofisher), 20 mM HEPES and 0.1 mM 2-mercaptoethanol. Preadipocytes were differentiated into mature adipocytes with 500 μM 3-isobutyl-1-methylxanine (Caymen Chemical Company, Ann Arbor, MI) and 1 μM Dethamexasone (Caymen Chemical Company) and maintained in DMEM supplemented with 10% FBS and 5 μg ml⁻¹ (ref. 56 (link)). Differentiated adipocytes were treated with 100 ng ml⁻¹ of recombinant mouse IL-1β (eBiosciences) for 6 h. For experiments involving co-treatment of adipocytes, cells were treated with 5 μM BMS345541 (Sigma-Aldrich), an NFκB inhibitor, 40 μM SP600125 (Sigma Aldrich) a JNK inhibitor or 500 μM metformin 1 h before treatment with recombinant mouse IL-1β.

For isolation of primary articular chondrocytes, cartilaginous femoral caps were removed from the femoral head of WT mice sacrificed at 2–3 weeks of age. Concretely, mice were sacrificed and disinfected in 75% ethyl alcohol prior to sampling. Then the femoral cartilaginous caps from bilateral femoral heads were digested by DMEM Nutrient Mixture F-12 (DMEM/F-12) medium (Thermo Fisher Scientific) containing 3 mg/mL Collagenase-D (Roche) and 1% penicillin/streptomycin (Thermo Fisher Scientific) for 6 hours at 37°C with intermittent percussion. Following digestion, the chondrocytes were harvested and cultured in complete DMEM/F-12 medium with 10% FBS. In this study, primary chondrocytes were not passaged and treatments were completed within 7 days of isolation from neonatal mice. For experiments in vitro, primary chondrocytes were exposed to recombinant mouse IL-1β (Sigma) at 10 ng/mL.

Acetylcholine assay kit, SB203580, recombinant mouse IL-1β, Krebs-ringer bicarbonate buffer (KRB) were purchased from Sigma (St. Louis, MO), Phospho-p38 MAP Kinase antibody was purchased from Cell Signaling Technology. Norepinephrine assay kit was ordered from Alpco.