Gemcitabine hydrochloride

Gemcitabine hydrochloride (Sigma, St. Louis, MO, USA) was resuspended in sterile normal saline at 10 mg/mL. Clodronate liposomes and PBS liposomes were purchased from clodronateliposomes.org (Haarlem, The Netherlands). The preparation was described earlier [33 (link)]. For in vitro experiments, Gemcitabine hydrochloride (Sigma, St. Louis, MO, USA), was used.

All tested compounds were dissolved in DMSO just before the experiment, and a calculated amount of drug solution was added to the cell growth medium to a final solvent concentration of 0.5%, which had no detectable effects on cell viability. Dichloroacetate (DCA), 2,2-dichloroacetophenone (DAP) and gemcitabine hydrochloride were purchased by Merck KGaA (Darmstadt, Germany).

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.