Fig. 4: Acidic lysosomal pH induces ferroptosis in senescent cells. | Nature Communications

Fig. 4: Acidic lysosomal pH induces ferroptosis in senescent cells.

From: Senescence-associated lysosomal dysfunction impairs cystine deprivation-induced lipid peroxidation and ferroptosis

Fig. 4

a Lysosomal pH of senescent TIG-3 (Irrad and Rep) cells after 24 h of treatment with or without 50 µM EN6, as measured by LysoSensor. b Measurement of free lysosomal ferrous iron (LysoRhoNox) and intracellular ferrous iron (FerroOrange) in senescent TIG-3 (Irrad and Rep) with or without 50 µM EN6 treatment. The box plot indicates the total intensity of LysoRhoNox staining. c Fluorescence images of lysosomes [LysoTracker (magenta)], lipid radicals [LipiRADICAL Green (green)], and nuclei [Hoechst 33342 (blue)] in senescent TIG-3 (Irrad and Rep) cells after treatment with 50 µM EN6. R represents the Pearson correlation coefficient between the signals of LysoTracker and LipiRADICAL Green. Scale bar, 50 µm. d Lipid peroxidation assessed by C11-BODIPY fluorescence in senescent TIG-3 cells (Irrad and Rep) with or without 2 µM erastin (Irrad), or 5 µM erastin (Rep) in the absence or presence of 50 µM EN6 for 12 h. The ratio of oxidized to reduced BODIPY (BODIPYox/BODIPYred) calculated from the median fluorescence intensity is shown. e Heatmap of peroxidized PC in senescent TIG-3 cells (Irrad) after 14 h of treatment with or without 5 µM erastin in the absence or presence of 50 µM EN6. f, g Cell viability of and LDH release by senescent TIG-3 (Irrad and Rep) cells after 24 h of treatment with or without 5 µM erastin in the absence or presence of 50 µM EN6 and with or without Fer-1 (500 nM), DFO (100 µM), Trolox (100 µM) or NAC (1 mM). h, i Senescent TIG-3 (Irrad and Rep) cells were infected with lentivirus encoding flag-tagged ATP6V1C2 or empty vector. After selection with puromycin, the cells were subjected to immunoblot analysis (h) or lysosomal pH analysis (i). j Viability of vector control or ATP6V1C2 overexpressing senescent TIG-3 cells (Irrad and Rep) after 24 h of treatment with 5 µM erastin alone or in combination with Fer-1 (500 nM), DFO (100 µM), Trolox (100 µM) or NAC (1 mM). k Mechanism of ferroptosis resistance in senescent cells. The data are presented as the mean ± s.d. of n  =  3 biological replicates (d, f, g, j). For (a, b, i) the box represents the interquartile range (IQR), with a horizontal line and a black point indicating the median and mean, respectively. Whiskers extend to the most extreme data points within 1.5 times the IQR from the quartiles. White points beyond this range represent outliers. Statistical analysis was performed using the Kruskal-Wallis test followed by Dunn’s test (a, b, i) and one-way ANOVA with Tukey’s test (d, f, g, j). Source data are provided as a Source Data file.

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