2.13. GPX4 Activity

GPX4 activity was evaluated by standard methods [29]. In detail, cell pellets were re-suspended in 0.75 mL lysis buffer (0.1 M Tris-HCl, 0.25 M sucrose, protease inhibitors, pH 7.5) and then sonicated and used in the test (0.1–0.2 mL of sample per test). Samples were mixed with the assay buffer (0.1 M Tris-HCl pH 7.8, 5 mM EDTA, 5 mM GSH, 0.1% (v/v) Triton X-100, 0.16 mM NADPH and 0.6 IU/mL Glutathione Reductase (GR)) and incubated for 5 min at 25 °C. Then the baseline was recorded at 340 nm for about 1 min and finally the enzymatic activity was started by adding phosphatidylcholine hydroperoxide (0.020 mM). The quantification of the activity was done on the basis of the net speed with which the absorbance decreases after the addition of the substrate (net speed = speed after the addition of the substrate − baseline speed).