Zalcitabine

Cells were treated with increasing concentrations of remdesivir (Cayman Chemical or MedChemExpress; purity > 98%; 0.8–400 µM), zidovudine (MedChemExpress, purity 98%; 0.8–1200 µM) or zalcitabine (Selleckchem, purity 100%; 2.4–1200 µM) for 24–120 h. zidovudine and zalcitabine are known for mitotoxic effects during HIV therapy (White 2001 (link)). zidovudine, but not zalcitabine, was shown to change the metabolism of H9c2 cells 6 days after treatment at 50 µM (Lund et al. 2007 (link)). Due to our own observation of zalcitabine reducing mitochondrial DNA in RPTEC/TERT1 cells after long term treatment (Fig. S5e), we compared remdesivir toxicity in cardiac cells or kidney cell lines to the acute toxicity of high doses zidovudine or zalcitabine, respectively. For positive control, cells were treated with high concentrations of dmso (up to 20%) or antimycin A (Sigma, up to 50 µM). ATP levels were determined as indicator of cell vitality of cells cultured to 70–100% confluence in 96-well plates by the CellTiter-Glo^® assay (promega) according to manufacturer’s instructions. Luminescence counts acquired by the synergy neo2 multi-mode reader using the software Gen5™ 3.10 (BioTek) were compared between treatment groups. Supernatants were stored to assess lactate concentrations and LDH-activity.

Zalcitabine (S1719), erastin (S7242), sulfasalazine (S1576), IKE (S8877), MitoQ (S8978), bafilomycin A1 (S1413), staurosporine (S1421), and CCCP (S6494) were purchased from Selleck Chemicals. MitoTEMPO (SML0737) was purchased from Sigma-Aldrich. The antibodies to STING1 (13647), TOMM20 (42406), MFN1 (14739), OPA1 (67589), PINK1 (6946), PRKN (4211), ACTB (3700), CALR (12238), and MAP1LC3B (3868) were purchased from Cell Signaling Technology. The antibody to MFN2 (ab205236) was purchased from Abcam. The antibody to mitHsp70 (MA3-028) was purchased from Thermo Fisher Scientific.

Etravirine, zalcitabine, tanespimycin, wortmannin, and calcium D-pantothenate were all purchased from Selleck (Shanghai, China) and ellipticine was purchased from MedChemExpress (MCE, Shanghai, China). All of these powders were dissolved in the appropriate solvent according to the manufacturer’s instructions.