Fig. 3: LDHB acts in parallel with GPX4 to suppress mitochondria-associated ferroptosis.

Immunoblot analysis of LDHB, GPX4, DHODH, LDHA, SLC7A11, and FSP1 in HT1080, MSTO-211H, A549, PANC-1 siRNAs cells, n = 3–6 independent repeats, and the statistical analysis was shown in Supplementary Fig. 3a, b (a). Colocalization analysis by immunofluorescence in HT1080 siRNAs cells after 72 h of transfection (b, scale bar, 20 µm). Images of microscopic analysis (c, 20x objective) and assessment of mitochondrial lipid peroxidation by flow cytometry in HT1080 sgNTC and sgGPX4 cells after 72 h of transfection with siRNAs, n = 3 independent replicates (d, e). Clonogenic assays of parental HT1080 (HT1080-P) and HT1080 cells treated with 4 µM RSL3 after ten cycles (HT1080-R) (f). Immunoblot analysis of LDHB, GPX4, DHODH, and LDHA in HT1080 parental and RSL3 resistant cells. The experiment was repeated three times, yielding consistent results (g). Cell viability of HT1080-P and HT1080-R cells after transfection with siRNAs treated with DMSO or RSL3 alone or in combination with 5 µM FER1 for 48 h, following pretreatment with vehicle, 5 µM FER1 for 24 h, n = 3 independent replicates (h). Immunoblot analysis of LDHB, GPX4, LDHA in HT1080 control and LDHB overexpression cells (i). Cell viability assays of HT1080 cells and HT1080 LDHB ORF cells treated with RSL3 with or without 5 µM FER1 for 48 h, following pretreatment with vehicle, 5 µM FER1 for 24 h. n = 3 independent replicates (j). Clonogenic assay of HT1080 siRNAs cells treated with DMSO or RSL3 alone or in combination with 2 mM glutathione reduced ethyl ester (GSH-mee) for 48 h after pretreatment with vehicle, 2 mM GSH-mee for 24 h (k). Tumor volume and weight of A549 sgNTC shCTRL (n = 12), sgNTC shLDHB (n = 11), sgGPX4 shCTRL (n = 12), sgGPX4 shLDHB (n = 12), sgGPX4 shLDHB treated with liproxstatin-1 (n = 12) xenograft tumors from different mice (l, m), tumor volume and weight of HT1080 sgNTC shCTRL (n = 10), sgNTC shLDHB (n = 10), sgGPX4 shCTRL (n = 10), sgGPX4 shLDHB (n = 12), sgGPX4 shLDHB treated with liproxstatin-1 (n = 12) xenograft tumors from different mice (o, p), 4-HNE expression of A549 and HT1080 sgNTC shCTRL, sgNTC shLDHB, sgGPX4 shCTRL, and sgGPX4 shLDHB xenograft tumors treated with or without liproxstatin-1, n = 10 or n = 12 different regions from different tumors from different mice respectively (n, q). Data were presented as mean ± SEM (l, o) or mean ± SD (e, h, j, m, n, p, q). Two-way ANOVA (h, j, l, o), Unpaired, two-tailed t-test (e, m, n, p, q). ns no significant difference, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Source data are provided as a Source Data file.