Nifedipine

Nifedipine, glibenclamide, acetylcholine, sodium pentobarbital, bovine serum albumin (BSA), HEPES, DL-Dithiothreitol (DTT), and 6-propyl-2-thiouracil (PTU) were purchased from Sigma (St. Louis, MO, USA); collagenase P, NADP disodium salt, ATP disodium salt, glucose-6-phosphate dehydrogenase from Roche (Roche Diagnostic, Mannheim, Germany), and all other reagent-grade chemicals from Merck (Darmstadt, Germany).
Stock solutions of Nifedipine and glibenclamide, were prepared in ethanol and dimethyl sulphoxide (DMSO) and the other substances were dissolved in H₂O or directly added into the incubation media.

For nifedipine treatment, 200 µM nifedipine (from a 500× stock; Sigma-Aldrich) in DMSO was added to the medium at stage 13. Embryos were fixed at stage 25.

NRK52E cells (2 × 10⁴ cells/well) were incubated in a 24-well plate for 16 h, and then treated with the nifedipine (Sigma, St. Louis, MO, USA) at different concentrations (0, 7.5, 15, or 30 μM) or with 150 μM oleic acid (Sigma, St. Louis, MO, USA) as a positive control for 48 h. The nifedipine dose was selected to correspond to internal levels in humans, and the positive control oleic acid dose was selected according to a preliminary 3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide (MTT) test. The cells were washed with phosphate-buffered saline (PBS) and incubated with 0.5 mg/mL MTT solution for 4 h at 37 °C prior to removing the culture medium. Dimethyl sulfoxide was then added and mixed for 5 min at 26 °C. Cell viability was determined by measuring the absorbance at 560 nm. Cell viability for each experimental group was calculated as a percentage of that of the control group.

Chondrocytes and BMMSCs were seeded into 12-well plates (SPL, Life Sciences, Gyeonggi-Do: South Korea) at a density of 20,000 cells/well in a complete medium. The next day, cells were divided into three treatment groups: control (with dimethyl sulfoxide (DMSO), which is a solvent for nifedipine and BayK8644 and was added the same amount as nifedipine and BayK8644 were), nifedipine (10 μM) and BayK8644 (10 μM) (Sigma-Aldrich, Hamburg, Germany). Cell proliferation was determined at days 1, 3, 5, 8, and 12 with cell counting kit -8 (CCK-8) (Dojindo, Munich, Germany) according to the manufacturer's instructions. Commercial CCK-8 kit allows to measure cell proliferation and cytotoxicity at once, by utilizing highly water-soluble tetrazolium salt. This salt is being reduced by living cell dehydrogenases and produces soluble orange formazan dye. This way the amount of the formazan dye generated by dehydrogenases in cells is directly proportional to the number of living cells. The medium was collected to 96 well plate (Orange Scientific, Braine-l'Alleud, Belgium) and absorbance at 450 nm was quantified with SpectraMaxvi3 spectrophotometer (Molecular Devices, San Jose, California, USA).

For drug response experiments, stock solutions of nifedipine (Sigma-Aldrich N7634), (±)-Bay K8644 (Tocris 1544) and ryanodine (Tocris 1329) were made in dimethyl sulfoxide (DMSO) at concentrations of 10, 20 and 25 mM, respectively. A stock solution of caffeine (Sigma-Aldrich N7634) was made in water at a concentration of 50 mM. Drug stocks were diluted in E3 medium at 28 ºC. After recording the basal images, 100 µL of the drugs were added to the wells, reaching a final concentration of 100 µM for nifedipine, (±)-Bay K 8644 and ryanodine, and 3 mM for caffeine. New sets of images were recorded after the incubation with the drugs, as indicated.