Fig. 5: Senescence and oxidative cell death are induced downstream of excessive localized H2O2 production.
From: Mitochondrial H2O2 release does not directly cause damage to chromosomal DNA
a Senescence associated-β-galactosidase staining of RPE1-hTERT-DAAOH2B and RPE1-hTERT-DAAOTOM20 cells. RPE1-hTERT-DAAOH2B cells become positive for the staining upon D-Ala addition. Staining is repeated 3 times, representative images are shown. Scale bar = 100 µm b Quantification of Senescence Associated-β-galactosidase positivity of RPE1-hTERT-DAAOH2B cells treated wilt L-Ala or D-Ala. Experiment was repeated 3 times, 2 images of each were quantified. Data are presented as mean -/+ SD. Statistical significance was determined by a two-sided unpaired t-test (***p ≤ 0.001, L-Ala vs. D-Ala = 0.0005). Source data are provided in the Source Data file. c Representative images of DAPI staining of RPE1-hTERT-DAAOH2B and RPE1-hTERT-DAAOTOM20 cells. RPE1-hTERT-DAAOH2B cells undergo chromatin restructuring in response to D-Ala treatment, demonstrating a phenotype reminiscent of senescence-associated heterochromatin foci (SAHFs). Scale bar = 20 µm. Phenotype has been observed in at least three independent experiments. d Quantification of IF stainings for senescence markers p21 and Lamin B1. Average signal per nucleus is shown. Induction of nuclear H2O2 results in a decrease in nuclear Lamin B1 signal and an increase in nuclear p21 intensity, both of which are markers of senescence. n = 2 biological independent repeats. For each repeat 3 images were analyzed per condition. Horizontal line represents median. Two-way Anova + Dunett’s multiple comparison test was performed (**p ≤ 0.01, ****p ≤ 0.0001, p21 DAAOTOM20 5 mM D-Ala = 0.0095, 10 mM D-ala = 0.0053). Source data are provided in the Source Data file. e Quantification of cell viability by PI exclusion in RPE1-hTERT-DAAOTOM20 cells. Dots represent 3 biological replicates. Per replicate, ~10.000 cells were analyzed by FACS. Data are presents as mean -/+ SD. Massive cell death occurs at 15 mM D-Ala, which can be rescued by ferroptosis inhibitors ferrostatin and liproxstatin. Two-way Anova + Dunett’s multiple comparison test was performed (****p ≤ 0.0001). Source data are provided in the Source Data file.
