Rankl

Human breast carcinoma cell lines MCF-7^GFP+Luc+ and MDA-MB-231^GFP+Luc+ (herein designated by MCF-7 and MDA-MB-231, respectively) were provided by Dr. Sérgio Dias (Instituto de Medicina Molecular, Lisbon, Portugal); and T47D cell line was provided by Dr. Phyllippe Clézardin (INSERM, Lyon, France). Cells were cultured under standard conditions, used at low passage number, and tested for Mycoplasma contamination. For continuous RANKL exposure experiments, medium was supplemented with 1 μg/ml RANKL (#11000457, Amgen Inc.) every 48 h for 6 weeks. Doubling time was calculated in GraphPad Prism 8 software, using the exponential growth equation with least squares regression fitting model.

All the cells were cultured in vitro at 37 °C, 5% CO₂. MSC growth medium comprised of Dulbecco’s Modified Eagle’s Medium (DMEM) (Lonza, UK) supplemented with 10% (v/v) foetal bovine serum (FBS) (Sigma-Aldrich, UK), L-glutamine final media concentration 2 mM (ThermoFisher Scientific, UK), and 100 units/ml penicillin-streptomycin (ThermoFisher Scientific, UK). MSC osteogenic medium comprised of MSC growth media supplemented with 100 nM dexamethasone (Sigma Aldrich, UK), 10 mM glycerolphosphate (Sigma Aldrich, UK) and 50 μg/ml L-ascorbic acid (Sigma Aldrich, UK). RAW growth medium comprised of α-MEM (Life Technologies, NZ) supplemented with 10% (v/v) FBS (Life Technologies, NZ), L-glutamine final media concentration 2 mM (Life Technologies, NZ) and 100 units/ml penicillin-streptomycin (Life Technologies, NZ). RAW cell differentiation medium comprised of growth media supplemented with 10 ng/ml RANK-L (Amgen). Mature osteoclast (MO) growth medium comprised of Earle’s MEM (ThermoFisher Scientific, NZ) supplemented with 10% (v/v) FBS, 100 units/ml penicillin-streptomycin and 0.1% 12 M HCL.