Fig. 2: Gpx4 is necessary for preventing lipid peroxidation and cell death induced by long-term cold exposure in hamster HapT1 cells.
From: Identification of genes supporting cold resistance of mammalian cells: lessons from a hibernator
a Immunoblot of Gpx4 and β-Actin proteins in parental (wild type; WT) and clonal HapT1 cells (#19, #21, #35) in which Gpx4 gene is disrupted. b The proportion of dead cells in WT or Gpx4-KO HapT1 cells during cold (4 °C) culture. c The proportion of dead cells in HT1080 and parental or Gpx4-KO HapT1 cells after 5 days of cold (4 °C) culture in the absence (non-treated; NT) or presence of 100 µM DFO, 1 µM ferrostatin-1 (Fer-1), 10 µM idebenone (Ide), 10 µM mitoquinol (MitoQ), 2 µM BAPTA-AM (One-way ANOVA with the Dunnett’s multiple comparison test compared to NT within each cell line, ***p < 0.001). d Oxidized lipid detection by BODIPY C11 ratio imaging in HT1080 and HapT1 cells (parental or Gpx4-KO) during cold (4 °C) culture. Scale bar = 200 µm. e Time-course changes of oxidized lipid level determined with BODIPY C11 ratio imaging in the presence or absence of 2 µM RSL3 or 1 µM Fer-1 N = 3. Plot is represented as mean ± s.d. f Detection of lipid peroxidation by LC–MS/MS analysis. The amount of mono-(left), di-(middle), tri-(right) oxidized phosphatidylethanolamine (PE38:4) in WT or Gpx4-KO HapT1 cells are shown (One-way ANOVA with the Tukey’s multiple comparison test, p < 0.05). g The proportion of dead cells in HapT1 after 5 or 7 days of cold (4 °C) culture in the absence or presence of 2 µM RSL3. Each bar corresponds to the condition shown in the upper panel wherein RSL3 was added only during the period indicated by black during the culture (One-way ANOVA with the Dunnett’s multiple comparison test, *p < 0.05, **p < 0.01).
