Immortalized HEK 293 Cell Culture

Immortalized human embryonic kidney (HEK 293) cells were commercially obtained from ATCC, catalogue Nr. CRL-1573. Cells were cultured in DMEM with GlutaMAX^TM (Gibco, Paisley, UK) or stable glutamine (PAN Biotech, Aidenbach, Germany) containing high glucose (25 mM) and 1 mM sodium pyruvate. The medium was supplemented with 50 μg/mL uridine (Sigma-Aldrich/Merck, Darmstadt, Germany), 10% heat-inactivated tetracycline-free FBS (PAN Biotech, Aidenbach, Germany), and 100 U/mL penicillin and streptomycin (Gibco, New York, NY, USA). The POLGexo^−/− cell line (p.D274A mutant) was obtained by CRISPR/Cas9 genome editing as described in Ref. [12 (link)]. The genotype was confirmed by Sanger sequencing (Figure S1).
Transient oxidative stress was induced on cells seeded at 90% confluence by applying H₂O₂ (Honeywell, Seelze, Germany) at concentrations of 0.5 or 1 mM. Viability was determined by adding 0.1% erythrosine B (Sigma-Aldrich, St. Louis, MO, USA) to cells suspended in 1× PBS (Gibco, Paisley, UK) and using a Neubauer hemocytometer (Paul Marienfeld, Lauda-Königshofen, Germany).