Supplementary Figure 3: Caspases are not involved in ferroptotic cell death.

(a) The pan caspase inhibitor Z-VAD-FMK (zvad) does not prevent cell death triggered by Gpx4 deletion. Knockout of Gpx4 was induced by adding 1 μM TAM in the presence of increasing zvad concentrations and cell viability was determined 72 h later. Data shown represents the mean ± s.d. of n = 3wells of a 96-well plate from a representative experiment performed independently three times. (b) Inducible Gpx4 deletion in Pfa-1 cells does not lead to caspase activation. Caspase activity was determined using CellEventTM Caspase-3/7 Green Detection Reagent (Life Technologies, Thermo Fisher Scientific) at different time points after KO induction. (c) Ferroptosis inducers do not increase caspase activity. Activities were determined in Pfa-1 cells after treatment with the different inducers at specific time points [TAM (1 μM – 48 h), BSO (10 μM – 48 h), erastin (1 μM – 12 h), RSL3 (0.5 μM – 3 h), staurosporine (Stauro; 0.2 μM – 6 h) and TNFα (10 ng ml⁻¹ – 9 h)]. (d) Class I FINs (BSO and erastin) decrease GSH levels, whereas the class II FINs RSL3 and Gpx4 do not impact on GSH levels. Total GSH levels were determined after treatment with the different inducers at specific time points [BSO (10 μM – 24 h), erastin (1 μM – 8 h), RSL3 (0.5 μM – 3 h) TAM (1 μM – 48 h). Data shown (b, c, d) represents the mean ± s.d. of n = 3 wells of a 6-well plate from a representative experiment performed independently two (b, c) or three (d) times. (e) Kinetics of cell death induced by ferroptosis-inducing agents and apoptosis-inducing agents in Pfa1 cells. Cells were treated with BSO (20 μM), erastin (2 μM), RSL3 (0.5 μM), TAM (1 μM), Staurosporine (0.5 μM) and TNFα (10 ng ml⁻¹). Cellular viability was assayed using AquaBluerTM. (f) Profile of rescue of Gpx4 proficient Pfa1 cells treated with apoptotic and ferroptotic cell death triggers. Cells were treated with the cell death inducers for the indicated times [TAM (1 μM – 72 h), erastin (1 μM – 24 h), RSL3 (0.5 μM – 24 h), TNFα (10 ng ml⁻¹ – 48 h) and Staurosporine (0.2 μM – 48 h)]. The rescue profile was performed using fixed concentrations of Z-VAD-FMK (zvad, 50 μM), Nec1 (25 μM), αToc (2 μM), Fer1 (0.2 μM) and DFO (10 μM), erastin (1 μM – 12 h), RSL3 (0.5 μM – 3 h), Staurosporine (0.2 μM – 6 h) and TNFα (10 ng ml⁻¹ – 9 h). (gh) Overexpression of active Gpx4 (WT), but not Gpx4-U46S in Gpx4-proficient Pfa1 cells, increases resistance to RSL3- and erastin-induced ferroptosis. Data shown represents the mean ± s.d. of n = 3 (e, f) or n = 4 wells (g, h) of a 96-well plate from a representative experiment performed independently three times.