Fig. 3: BAX and BAK macropores mediate mtDNA release and the SASP in senescent cells. | Nature

Fig. 3: BAX and BAK macropores mediate mtDNA release and the SASP in senescent cells.

From: Apoptotic stress causes mtDNA release during senescence and drives the SASP

Fig. 3

a, CRISPR–Cas9 gene editing was used to generate human fibroblasts deficient in BAX and BAK (BAX−/− BAK−/−). Western blot showing successful CRISPR–Cas9-mediated deletion of BAK and BAX in proliferating and senescent (IR) cells. BAX/BAKCRISPR indicates cells negative for BAX and BAK. Western blot is representative of n = 3 independent experiments. b, The mean number of cytosolic DNA foci in proliferating and senescent BAX−/−BAK−/− cells. n = 4 independent experiments. c, Column-clustered heat map of SASP genes that are differentially expressed in senescence and rescued by deletion of BAX and BAK. The colour intensity represents the column Z-score; red and blue indicate high and low expression, respectively. d, The levels of secreted cytokines in proliferating and senescent empty vector (EV) and BAX−/−BAK−/− cells. n = 6 independent experiments. e, mRNA levels of CDKN2A (n = 4 independent experiments) and CDKN1A (n = 6 independent experiments) in proliferating and senescent EV and BAX−/−BAK−/− cells. f,g, The percentage p16INK4a-positive (f) and p21-positive (g) proliferating and senescent EV and BAX−/−BAK−/− cells. n = 7 independent experiments. h,i, Quantification of the percentage of senescence-associated β-galactosidase (Sen-β-Gal)-positive cells (h) and the mean number of γH2AX foci in proliferating and senescent EV and BAX−/−BAK−/− cells (i). n = 4 independent experiments. j, Western blot analysis of lamin B1 and HMGB1 levels in proliferating and senescent EV and BAX−/−BAK−/− cells. Representation of n = 3 independent experiments. k, The percentage of Ki-67-positive proliferating and senescent EV and BAX−/−BAK−/− cells. n = 4 independent experiments. l, Column-clustered heat map of proliferation genes that are differentially expressed in senescent cells and are not rescued by deletion of BAX and BAK. The colour intensity represents the column Z-score. m, Representative microscopy images of Ki-67 (red) and γH2AX (green) (top); p21 (red) and p16INK4a (green) (blue is DAPI) (middle); and Sen-β-Gal (bottom) in proliferating and senescent EV and BAX−/−BAK−/− cells. Scale bars, 100 µm. Representative images of n = 4 independent experiments. Data are mean ± s.e.m. Statistical significance was assessed using one-way ANOVA followed by Tukey’s multiple-comparison test (b, ei and k). Individual data points are from biological replicates. Gel source data for j are provided in Supplementary Fig. 1.

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