Intracellular Glutathione Measurement Protocol

Total intracellular glutathione measurements were performed as previously described [40 (link)]. Briefly, 5 × 10⁵ H1299p53^R273H Cas9 and H1299p53^R273H-Casp2^−/− cells with and without 2 µM erastin treatment for 12 h were homogenized in ice-cold 10 mM HCl. Proteins were precipitated by adding 5-sulfosalicylic acid (Sigma-Aldrich) to a final concentration of 1%. Samples were centrifuged to remove precipitates and supernatants were collected and stored at –20 °C until analysis. Samples were incubated in the presence of 5,5’-dithio-bis-[2-nitrobenzoic acid] (DTNB, 0.73 mM) (Sigma-Aldrich), EDTA (4 mM) (Sigma-Aldrich), dihydronicotinamide- adenine dinucleotide phosphate (NADPH, 0.24 mM) (Sigma-Aldrich), 110 mM sodium phosphate (NaH2PO4) buffer (pH 7.4) and glutathione reductase (GR; Sigma-Aldrich) from baker’s yeast (1.2 U/ml). The absorbance at 412 nm was measured every 15 sec for 5 min on a Cytation 3 Imaging Reader (BioTek). The total concentration of GSH + GSSG was calculated based on a GSH standard curve.