Uk5099

For Alizarin Red Staining for calcium deposition, HAT7 cells were cultured in 24-well plastic plates coated with collagen type I (#638-00781, Nitta Gelatin Co., Osaka, Japan) at confluence in calcification induction medium; DMEM/F12 supplemented with 10% FBS, dexamethasone (10 nM), CaCl₂ (final concentration 2.1 mM) for 7 days under normoxia (21% O₂) or hypoxia (5% O₂), or for 5 days with UK-5099 (#S5317, Selleckchem, Randnor, PA, United States). The culture supernatant in the wells was removed, and the cells were washed with PBS, fixed with 4% PFA, and then washed three times with distilled water. Next, a 1.0% Alizarin Red S (#A5533, Sigma-Aldrich) stain was added, and the mixture was allowed to stand at room temperature for 30 min. The cells were then washed three times with PBS. The collagen gels with the cells were placed on the prepared slide and then dried at 37°C for 1 h. After drying, the gels were observed.

Hyperthermia was applied in a 42 °C water bath at 5% CO₂ for one hour, unless specified otherwise. N-acetyl-l-cysteine, Trolox, DMSO, MitoQ, and tetrathiomolybdate (all from Merck) were present from 6 h before until 24 h after the hyperthermia treatment. Elesclomol (Selleck, Houston, TX, USA), epirubicin hydrochloride, mitomycin C, gemcitabine hydrochloride, menadione, and hydrogen peroxide (all from Merck) were added 5 min prior to hyperthermia and removed 5 min afterwards. Thiram, TMT, Zn-pyrithione (Sigma, St. Louis, MO, USA), 8HQ, clioquinol, and GTSM-Cu (Medchem Express, Monmouth Junction, NJ, USA) were added 30 min prior to hyperthermia and removed 24 h after. Elesclomol was used in combination with copper(I) chloride (Merck) at an equimolar ratio unless stated otherwise. In cell viability experiments involving mono-exposure to copper, copper(I) chloride was added 5 min before hyperthermia and left until the end of the experiment. Antimycin-A (Selleck) and FCCP (Abcam, Cambridge, UK) were added 1 h prior to hyperthermia and left until the end of the experiment. DMNQ (Medchem Express) and paraquat dichloride (Merck) were added 5 min prior to hyperthermia and left until the end of the experiment. BPTES, etomoxir, and UK5099 (all from Selleck) were added 4–6 h prior to hyperthermia and removed after 24 h.