Clioquinol

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.

The chemicals, such as ZnCl₂ (Cat#10023818), Zn(CH₃COO)₂ (Cat#30192618), and ZnSO₄ (Cat#10024018) were obtained from Sinopharm Chemical Reagent Co, Ltd. (Shanghai, China). Compounds, 48/80 (Cat#C2313) and chloroquine (Cat#C6628) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Pyrithione (Cat# HY-B1747-10 mg) and Clioquinol (Cat# HY-14603) were obtained from MCE (MedChemExpress, New Jersey, USA). TPEN (Cat#4309/100), HC030031 (Cat#2896), A967079 (Cat#4716), capsazepine (CPZ, Cat#0464), HC067047 (Cat#4100), and U0126 (Cat#U120) were obtained from Tocris (Bristol, UK). Morphine hydrochloride was obtained from China Northeast Pharmaceutical Group Shenyang No. 1 Pharmaceutical CO., Ltd (Shenyang City, Liaoning Province, China). Naloxone hydrochloride was obtained from China Sinopharm Group Guorui Pharmaceutical CO., Ltd (Huainan City, Anhui Province, China). TPEN, pyrithione, and Clioquinol were dissolved in 10% dimethyl sulfoxide (DMSO). Unless otherwise specified, other reagents are dissolved in sterile saline.