Apoptotic stress causes mtDNA release during senescence and drives the SASP methods
Aim. Evidence-backed execution summary for Apoptotic stress causes mtDNA release during senescence and drives the SASP methods from Apoptotic stress causes mtDNA release during senescence and drives the SASP.
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mouse
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Methods
reagent used in the protocol.
- Use
- Human embryonic lung MRC5 fibroblasts (ATCC) and IMR90 fibroblasts (ATCC) were grown in Dulbecco's modified Eagle's medium (Sigma-Aldrich, D5796) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U ml -1 penicillin, 100 µg ml -1 streptomycin and...
Methods
reagent used in the protocol.
- Use
- Stress-induced senescence was achieved by exposing cells to X-ray irradiation at 10 Gy (MAFs) or 20 Gy (human fibroblasts). Replicative senescence was achieved by serially passaging cells until they reached their replicative potential and performed less than 0.5 population doublings for at least 4...
Methods
reagent used in the protocol.
- Use
- 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 (S...
Methods
reagent used in the protocol.
- Use
- For therapy-induced senescence, cells were treated with either 250 nM of doxorubicin or 50 µM etoposide for 24 h. After 24 h, the culture medium was refreshed. Cells were maintained in culture and collected at days 10 and 8 after treatment, respectively.
Methods
reagent used in the protocol.
- Use
- To induce mitochondrial fragmentation, cells were treated with 12.5 µM CCCP at days 2 and 3 after irradiation. At day 4, the cell culture medium was refreshed, and the cells were maintained in culture until day 10 after irradiation. Cells were collected at day 10 for analysis.
Methods
reagent used in the protocol.
- Use
- The plasmids pFU-GEV16 and pF5XUAS-UL12.5, containing HSV-1 UL12.5, were a gift from G. Hacker. MRC5-UL12.5 cells were generated as described previously. In brief, MRC5 fibroblasts were first transfected with the pFU-GEV16 construct (expression vector that contains the transcriptional activator). Cells were selecte...
FPLC
reagent used in the protocol.
- Use
- 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) glycer...
qPCR
reagent used in the protocol.
- Use
- For bone, osteocyte-enriched cell samples were generated as described previously. The samples were homogenized in QIAzol Lysis Reagent (Qiagen) and immediately stored at -80 °C. Total RNA was extracted using QIAzol Lysis Reagent followed by purification using RNeasy Mini Columns (Qiagen). For the l...
qPCR
For bone, osteocyte-enriched cell samples were generated as described previously. The samples were homogenized in QIAzol Lysis Reagent (Qiagen) and immediately stored at -80 °C. Total RNA was extracted using QIAzol Lysis Reagent followed by purification using RNeasy Mini Columns (Qiagen). For the l...
- Use
- For bone, osteocyte-enriched cell samples were generated as described previously. The samples were homogenized in QIAzol Lysis Reagent (Qiagen) and immediately stored at -80 °C. Total RNA was extracted using QIAzol Lysis Reagent followed by purification using RNeasy Mini Columns (Qiagen). For the l...
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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...
Measurement outputs
What raw and processed outputs should exist?
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...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
Our observations, as well as our recent research demonstrating a key role for mitochondrial dynamics in regulating miMOMP, led us to hypothesize that mitochondrial hyperfusion...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
For cytotoxicity analysis, cells were treated with ABT-263 at the indicated concentrations for 24 h before assessment of cytotoxicity.
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
X-ray-irradiated cells were treated with 10 µM eltrombopag (provided by E. Gavathiotis) for 10 days (treatment was added immediately after irradiation). The tre...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
Analysis plan
How should the outputs become interpretable results?
Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
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.
from paperScoring or quantification
Quantify the primary readouts for this experiment: 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...; Our observations, as well as our recent research demonstrating a key role for mitochondrial dynamics in regulating miMOMP, led us to hypothesize that mitochondrial hyperfusion...; For cytotoxicity analysis, cells were treated with ABT-263 at the indicated concentrations for 24 h before assessment of cytotoxicity.; X-ray-irradiated cells were treated with 10 µM eltrombopag (provided by E. Gavathiotis) for 10 days (treatment was added immediately after irradiation). The tre....
from paperStatistical comparison
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...; Our observations, as well as our recent research demonstrating a key role for mitochondrial dynamics in regulating miMOMP, led us to hypothesize that mitochondrial hyperfusion...
from paperReporting output
Report representative outputs alongside summary comparisons for 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..., Our observations, as well as our recent research demonstrating a key role for mitochondrial dynamics in regulating miMOMP, led us to hypothesize that mitochondrial hyperfusion..., For cytotoxicity analysis, cells were treated with ABT-263 at the indicated concentrations for 24 h before assessment of cytotoxicity., X-ray-irradiated cells were treated with 10 µM eltrombopag (provided by E. Gavathiotis) for 10 days (treatment was added immediately after irradiation). The tre....
inferred from protocolStructured statistical methods
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...; Our observations, as well as our recent research demonstrating a key role for mitochondrial dynamics in regulating miMOMP, led us to hypothesize that mitochondrial hyperfusion...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (4)
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.
Machine-readable layer
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