Risedronate

Thiazolyl blue tetrazolium bromide and risedronate were purchased from Sigma‐Aldrich Inc. The TRAP staining kit was purchased from Cosmo Bio Co. Dulbecco's Modified Eagle Medium (DMEM), hematoxylin and eosin (H&E), 4′,6‐Diamidino‐2‐Phenylindole (DAPI), as well as p38 MAPK beta and AP1/JUN/RUNX2/OCN antibodies were purchased from Thermo Fisher Scientific Inc. Integrated DNA Technologies provided the primers (NFATc1, TRAP, GAPDH (Mouse), ALP, OPG, and GAPDH (Human)). AMRESCO provided a phenol‐free total RNA purification kit.

In total, 1 × 10⁵ clone 1C116 or 2C21 cells, were cultured with each material (concentration ranges are described below) or vehicle control (dimethyl sulfoxide: DMSO) and 20 ng/mL PMA in 96-well U-bottom tissue culture plates in 200 μL of CCM for 24 h. The materials were 0–100 μg/mL flavonoids (quercetin, isoquercitrin, hyperoside, galangin, kaempferol, morin, myricetin, hesperetin, hesperidin, acacetin, linarin, isorhoifolin, (all purchased from Extrasynthese, Lyon, France)), 0–1000 μM IPP (Sigma-Aldrich), 0–2000 μM risedronate, 0–33.5 μM methylamine (Sigma-Aldrich), 0–33.5 μM ethylamine (Sigma-Aldrich) and 1 μg/mL purified anti-human CD3 mAb (OKT3: Thermo Fisher Scientific). After the incubation, the culture supernatant was corrected, and the concentration of IL-2 was analyzed using an ELISA kit (BD Biosciense).

Alendronate, zoledronate, and risedronate (Sigma-Aldrich, St. Louis, MO, USA) were resuspended and diluted at 1 μM and 5 μM in DMEM 10% FBS and 1% penicillin/streptomycin. Cells were seeded in 96-well plates, transfected 24 h later and media was replaced with BP-containing media 24 h post-transfection. Viability and enzymatic assays were performed 48 h after BP exposure. Enzymatic activity was measured by the conversion of a CYP1A1 proluciferin substrate into a luciferin product detectable by luminescence using a P450-Glo Assay (Promega, Madison, WI, USA). The non-lytic assay was performed directly in the cultured cells following the commercial protocol. Viability assays were performed by rinsing the wells in PBS and adding 100 µL/well of MTT (Sigma-Aldrich, St. Louis, MO, USA) diluted 1:10 in DMEM. Cells were incubated for 3 h at 37 °C. Then, the media was aspired and 100 µL/well of DMSO (Merck-Millipore, Burlington, MA, USA) were added. Plates were shaken at 150 rpm for 5 min and media was transferred to a transparent plate for its quantification in a Modulus Microplate (Thermo Fisher Scientific, Waltham, MA, USA) at 560 nm.