Apoptotic stress causes mtDNA release during senescence and drives the SASP methods
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BAX and BAK promote the SASP in vivo
To investigate a role of BAX and BAK in senescence in vivo, we used Bax fl/fl Bak -/- mice and performed tail-vein injection with AAV-TBG-Cre virus to delete Bax in the liver. We exposed mice to 4 Gy of ionizing irradiation (IR), a dose that was previously shown to induce senescence and the SASP in the liver, (Fig. ). The absence of BAX in the liver was confirmed using immunohistochemistry (Fig. ). We next characterized the mRNA expression of different pro-inflammatory components 6 days after IR and found that, while these were significantly increased in Bak -/- mice, deletion of both Bax and Bak suppressed their induction (Fig. ). Nonetheless, when we analysed the senescence marker telomere-associated foci, which denotes co-localization between the DNA damage response protein γH2A.X and telomeres, we found that it was induced by irradiation i...
Methods
For induction of miMOMP, proliferating cells were treated with 2.5 µM ABT-737 (Abcam, ab141336) for 9 and 23 days. Treatment was refreshed every 48-72 h. For inhibition of the MPTP, cells were irradiated with 20 Gy X-ray irradiation and treated with 1 µM cyclosporin A (Sigma-Aldrich, SML1575) for 10 days. For BAX inhibition, MRC5 fibroblasts were irradiated with 20 Gy X-ray irradiation and treated with BAX inhibitor (BAI1) (Adooq Biosciences, A15335) at the indicated concentrations. Cyclosporin A and BAI1 were added straight after irradiation and maintained in the cell culture medium for 10 days (refreshed every 48-72 h).
FPLC
Cells grown in 150 cm 2 flasks were trypsinized and pooled to obtain sufficient material for the assay. After PBS washes, cells were centrifuged at 900 g for 5 min. Cell pellets were then lysed using CHAPS lysis buffer (1% (w/v) CHAPS, 20 mM HEPES at pH 7.4, 150 mM NaCl, 1% (v/v) glycerol, 1 mM PMSF, 10 µg ml -1 leupeptin, 10 µg ml -1 pepstatin, 100 mM NaF, 10 mM sodium pyrophosphate, 1 mM sodium vanadate and 20 nM microcystin) for 30 min at 4 °C. The samples were diluted to contain 10 mg ml -1 of protein and 200 µl was injected onto a Superdex S200 size-exclusion column. Twenty 500 µl fractions were collected. Protein precipitation using trichloroacetic acid (TCA) was then performed. In brief, the samples were incubated wit...
Immunohistochemistry
Formalin-fixed paraffin-embedded tissue sections (5 µm) were deparaffinized in xylene (3 times for 3 min each) and hydrated using 100% ethanol (twice for 5 min), 70% ethanol (5 min) and distilled water (twice for 3 min each). Antigen retrieval was done by heating the sections to 98 °C in citrate buffer pH 6.0 (Agilent-Dako, S236984) for 30 min. The slides were allowed to cool down for 30 min and were then rinsed in TBS twice for 5 min. Tissue sections were blocked with NGS (Agilent-Dako, X090710-8) for 30 min at room temperature followed by primary antibody incubation overnight at 4 °C. The sections were washed in TBS and incubated with peroxidase block (Agilent-Dako, K500711) for 30 min at room temperature. HRP-conjugated secondary antibody was then applied for 30 min at room temperatur...
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To investigate a role of BAX and BAK in senescence in vivo, we used Bax fl/fl Bak -/- mice and performed tail-vein injection with AAV-TBG-Cre virus to delete Bax in the liver. We exposed mice to 4 Gy of ionizing irradiation (IR), a dose that was previously shown to induce senescence and the SASP in the liver, (Fig. ). The absence of BAX in the liver was confirmed using immunohistochemistry (Fig. ). We next characterized the mRNA expression of different pro-inflammatory components 6 days after IR and found that, while these were significantly increased in Bak -/- mice, deletion of both Bax and Bak suppressed their induction (Fig. ). Nonetheless, when we analysed the senescence marker telomere-associated foci, which denotes co-localization between the DNA damage response protein γH2A.X and telomeres, we found that it was induced by irradiation irrespectively of the genotype (Supplementary Fig. ). IR did not affect the percentage of caspase-3 high liver cells, which suggests that senescence-rather than apoptosis-was induced under these conditions (Supplementary Fig. ). To further test the hypothesis that BAX and BAK regulate the...
For induction of miMOMP, proliferating cells were treated with 2.5 µM ABT-737 (Abcam, ab141336) for 9 and 23 days. Treatment was refreshed every 48-72 h. For inhibition of the MPTP, cells were irradiated with 20 Gy X-ray irradiation and treated with 1 µM cyclosporin A (Sigma-Aldrich, SML1575) for 10 days. For BAX inhibition, MRC5 fibroblasts were irradiated with 20 Gy X-ray irradiation and treated with BAX inhibitor (BAI1) (Adooq Biosciences, A15335) at the indicated concentrations. Cyclosporin A and BAI1 were added straight after irradiation and maintained in the cell culture medium for 10 days (refreshed every 48-72 h).
Cells grown in 150 cm 2 flasks were trypsinized and pooled to obtain sufficient material for the assay. After PBS washes, cells were centrifuged at 900 g for 5 min. Cell pellets were then lysed using CHAPS lysis buffer (1% (w/v) CHAPS, 20 mM HEPES at pH 7.4, 150 mM NaCl, 1% (v/v) glycerol, 1 mM PMSF, 10 µg ml -1 leupeptin, 10 µg ml -1 pepstatin, 100 mM NaF, 10 mM sodium pyrophosphate, 1 mM sodium vanadate and 20 nM microcystin) for 30 min at 4 °C. The samples were diluted to contain 10 mg ml -1 of protein and 200 µl was injected onto a Superdex S200 size-exclusion column. Twenty 500 µl fractions were collected. Protein precipitation using trichloroacetic acid (TCA) was then performed. In brief, the samples were incubated with one-tenth sample volume of 10% Triton X-100 and one-fifth sample volume of 100% ice-cold TCA for 20 min on ice. The samples were then centrifuged for 5 min at 800 g at 4 °C, the supernatant was discarded and the pellet was washed once with 1 ml ice-cold 10% TCA and twi...
Formalin-fixed paraffin-embedded tissue sections (5 µm) were deparaffinized in xylene (3 times for 3 min each) and hydrated using 100% ethanol (twice for 5 min), 70% ethanol (5 min) and distilled water (twice for 3 min each). Antigen retrieval was done by heating the sections to 98 °C in citrate buffer pH 6.0 (Agilent-Dako, S236984) for 30 min. The slides were allowed to cool down for 30 min and were then rinsed in TBS twice for 5 min. Tissue sections were blocked with NGS (Agilent-Dako, X090710-8) for 30 min at room temperature followed by primary antibody incubation overnight at 4 °C. The sections were washed in TBS and incubated with peroxidase block (Agilent-Dako, K500711) for 30 min at room temperature. HRP-conjugated secondary antibody was then applied for 30 min at room temperature, followed by TBS washes and DAB incubation for 5-10 min. The sections were counterstained with haematoxylin according to a standard procedure. A list of the antibodies used is provided in Supplementary Table.
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