Rpe 1

A431 (CRL-1555), EA.hy926 (CRL-2922), A498 (HTB-44) and CFPAC-1 (CRL-1918), Jurkat (TIB-152) and RPE-1 (CRL-4000) cells were obtained from the ATCC. They were grown in DMEM (A431, EA.hy926, A498 and CFPAC-1), RPMI medium (Jurkat) or DMEM F12 (RPE-1), supplemented with 10% FBS, and were maintained at 37 °C in a humidified atmosphere of 95% air/5% CO₂. Mycoplasma testing was regularly performed.

The human cell lines RPE-1, U2OS and HEK293 were obtained from ATCC, Manassas, VA, USA and cultured in DMEM/F12 (RPE-1) or DMEM (U2OS and HEK293) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 µg/mL streptomycin (herein referred to as complete medium, CM) (all from Sigma-Aldrich, St. Louis, MO, USA) at 37 °C and 5% CO₂. The cells have been authenticated and regularly tested for mycoplasma contamination. Depending on the assay, cells were seeded one or two days prior to the experiments. Cells were starved for amino acids by incubation in Earle’s balanced salt solution (EBSS, #24010043, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% dialyzed FBS (EBSS/FBS, dialysis tube cutoff 3.5 kDa). For studies using inhibitors or antioxidants, the cells were pre-treated for either 1.5 h (BIRB 796 and U0126, both 10 µM), 1 h (NAC, 3 mM; GSH, 10 mM), or 30 min (SB203580, SB202190 and JNK-IN-8, 3 µM; PF-3644022, 2.5 µM) before addition of PBCA. To assess cell viability after PBCA treatment, cellular ATP was measured as an indicator of metabolically active cells using CellTiter-Glo^® Luminescent Cell Viability Assay (#G7571, Promega, Madison, WI, USA). Luminescence was measured by a Synergy2 plate reader (BioTek Instruments Inc., Winooski, VT, USA).

Human HEK293, U2OS, RPE-1, and HeLa cells were obtained from ATCC. All cell lines were cultured in DMEM containing 10% FBS and 5% Penicillin/Streptomycin. All cell lines were regularly screened for mycoplasma using a MycoAlert™ Mycoplasma Detection Kit.