Fig. 1: Enhanced GPx4 expression in mtDNA-depleted SK-Hep1 ρ⁰ cells and resistance to cell death by ferroptosis inducers.

A mRNA expression of GPx4 in GSK-Hep1 ρ⁺ cells and SK-Hep1 ρ⁰ cells was examined by real-time RT-PCR (left) and RT-PCR (right). B GPx4 protein expression in SK-Hep1 ρ⁺ cells and SK-Hep1 ρ⁰ cells were determined by immunoblot analysis using the indicated antibodies. C Expression of different isoforms of GPx4 in SK-Hep1 ρ⁺ cells and ρ⁰ cells were studied by real-time RT-PCR. D GPx4 protein levels in the cytoplasmic and mitochondrial fractions were determined using immunoblot analysis. Purity of the cytoplasmic and mitochondrial fractions was confirmed by immunoblot analysis using anti-α-tubulin and -COXIV antibodies, respectively. E SK-Hep1 ρ⁺ cells and ρ⁰ cells were treated with 2.5 and 5 µM erastin or 0.1 and 1.0 µM RSL3 for 24 h. Cell death was measured using LDH release assay. F SK-Hep1 ρ⁺ cells and ρ⁰ cells were treated with 5 μM erastin or 0.1 μM RSL3 for 24 h in the presence or absence of 2.5 µM Fer-1 (Ferrostatin-1), 0.1 µM Lip-1 (Liproxstatin-1), 10 µM ZIL (Zileuton), 50 µM DFO (Deferoxamine), 50 µM Trolox or 50 µM z-VAD (z-VAD-FMK). Cell death was determined using LDH release assay. G Nrf2 protein level in SK-Hep1 ρ⁺ cells and ρ⁰ cells was determined using immunoblot analysis. H GPx4 protein level in SK-Hep1 ρ⁰ cell extract was determined using immunoblot analysis with or without treatment with 10 µM ML385, an Nrf2 inhibitor, for 24 h. I SK-Hep1 ρ⁺ and ρ⁰ cell death after treatment with 5 µM erastin for 24 h was determined using LDH release assay with or without 10 µM ML385 or 2.5 µM Fer-1 pretreatment for 1 h. Data represent means ± SD from three independent experiments. Data were analyzed by two-tailed Student’s t test (A, C, E, ρ⁺ and ρ⁰ cell comparisons in F) or one-way ANOVA with Tukey’s multiple comparison test (F except ρ⁺ and ρ⁰ cell comparisons, I). (CON, control) (*p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant) (Uncropped immunoblots can be found in Fig. S2).