Gm6001

RC6 spheroids were generated using the hanging drop method with methylcellulose, as previously described [17 (link)]. For 3D invasion assay, 25 μl of spheroids was embedded in hydrogel mixture (17.85 μl collagen type I; 0.45 μl 1 M NaOH; 5 μl 5x DMEM; 1.7 μl dH₂O), placed on top of 50 μl of hydrogel, and treated with 10 μM CoQ10, 250 μM TMZ, their combination, or 50 μM GM6001 (Selleckchem, TX, USA). Control without treatment was included. After 24 h, spheroids were stained with CAM (1 : 1000) and PI (1 : 500) in order to visualize spheroids and test viability. Images of spheroids were taken using a Nikon Eclipse Ti microscope equipped with a C1 modular confocal microscope system, at 20x magnification. The size of spheroids was analyzed using Fiji® software. At least 10 spheroids were analyzed per experiment.

G-749, MG132, z-VAD-FMK, GM6001 and Necrostatin-1 were purchased from Selleckchem (Houston, TX, United States). Hydroxypropyl-β-cyclodextrin, cycloheximide, crystal violet and chloroquine were purchased from Sigma Chemical Co. (St. Louis, MO, United States). Compound E was purchased from MedChemExpress (Monmouth Junction, NJ, United States). The antibodies used in this study are shown in Supplementary Table S1.

Peripheral blood mononuclear cells were isolated from human leucocyte cones (NHS Blood and Transplant, Bristol, UK; anonymous donors) by density gradient centrifugation. CD14 + monocytes were positively selected (MACS CD14 + microbeads; Miltenyi Biotech, Surrey, UK) and seeded at 0.25 × 10⁶ cells/well into 96-well plates containing dentine discs, 1 × 10⁶ cells/well into 48-well plates containing cartilage pieces, or 1 × 10⁶ cells/well into 24-well plates. After overnight adhesion, dentine and cartilage was transferred to new wells. Osteoclast differentiation was induced with 25 ng/ml M-CSF (R&D Systems, Abingdon, UK) and 50 ng/ml RANKL (Peprotech, London, UK) in α-MEM containing 10% FBS, 50 IU/ml penicillin, 50 μg/ml streptomycin sulphate and 2 mM L-glutamine. Differentiation medium was refreshed every 2–3 days for 10–13 days. For inhibitor experiments, mature osteoclasts were treated with bafilomycin (Cell Signalling Technology, Hitchin, UK), E64 (Cambridge Bioscience, Cambridge, UK), GM6001 (Selleckchem, Munich, Germany) or recombinant human TIMP1 (R&D Systems).

MLN8237 and GM6001 were purchased from Selleck Chemicals. Cisplatin was purchased from Sigma-Aldrich. All chemicals were dissolved in dimethyl sulfoxide (DMSO) for in vitro studies.