Supplementary Figure 2: Genetic loss of Alox15 does not rescue cell death and ARF induced by Gpx4 inactivation.

(a) TAM-inducible Gpx4 deletion in lung fibroblasts derived from CreERT2;Gpx4fl/fl/Alox15^−/− mice (PZL cells). (b) PZ (Alox15-proficient) and PZL (Alox15-deficient) lung fibroblasts can be rescued from Gpx4 deletion-induced cell death by lipophilic (αToc), but not by hydrophilic antioxidants (NAC, N-acetylcysteine). Gpx4 knockout was induced by the addition of 1 μM TAM in the presence of increasing concentrations of the antioxidants; cell viability was measured 72 h later. (c,d) Knockdown of Alox5 delays cell death upon Gpx4 deletion only in cells additionally lacking Alox15 (PZL) (d), but not in cells proficient for Alox15 (PZ) (c). Cell death was monitored at different time points after KO induction using AquaBluerTM. Knockdown efficiency was monitored by quantitative PCR. Data shown represents the mean ± s.d. of n = 3 wells (b, c, d) of a 96-well plate (or 6-well plate for RNA isolation) from a representative experiment performed independently three times, ANOVA (^∗p < 0.05). (e) Histological analysis of kidneys of TAM-treated CreERT2;Gpx4^fl/fl/Alox15^−/− mice revealed widespread tubular cell death, interstitial edema, and proteinaceous casts in distal tubules like TAM-treated CreERT2;Gpx4^fl/fl/Alox15^+/+ mice (scale bars 100 μm) (Periodic Acid-Schiff Stain, PAS). (f) Electron micrographs confirmed the severe destruction of renal tubule cells in symptomatic CreERT2;Gpx4^fl/fl/Alox15^+/+ mice (scale bars 10 μm). (g) Staining against F4/80, a 160 kD glycoprotein expressed by murine macrophages, revealed interstitial inflammatory cell infiltrates in response to massive tissue injury in symptomatic Gpx4 null kidneys (CreERT2;Gpx4^fl/fl). Staining against active caspase-3 (a-Casp-3) showed faint staining in kidney tissue of Gpx4 knockout mice.