Science Note: Senescence

Apoptotic stress causes mtDNA release during senescence and drives the SASP
Stella Victorelli, et. al., Nature, 2023.
Apoptosis is triggered by widespread mitochondrial outer membrane permeabilization (MOMP), whereas in cellular senescence, sublethal minority MOMP occurs in a subset of mitochondria, releasing mtDNA into the cytosol and inducing SASP-driven inflammation.

Highlighted technique: When apoptotic stress increases the permeability of the mitochondrial outer membrane, cytochrome c is released into the cytoplasm, making it a key indicator of MOMP. In this study, MOMP is assessed by co-staining the mitochondrial outer membrane protein TOM20 and cytochrome c, followed by high-resolution analysis.

Mitochondrial fatty acid oxidation drives senescence
Shota Yamauchi, et. al., Science Advances, 2024.
DNA damage triggers the phosphorylation of BNIP3, a mitochondrial membrane protein, leading to an increase in cristae and enhanced FAO. This, in turn, promotes histone acetylation and induces the expression of the senescence marker p16.

Highlighted technique: This study assessed senescence using multiple markers, including p16 expression and SA-βgal activity. While other markers changed, γH2AX did not, reinforcing the widely accepted notion that no single marker defines senescence. Thus, multiple indicators are needed to assess senescence.

Senescent glia link mitochondrial dysfunction and lipid accumulation
China Byrnsn, et. al., Nature, 2024.
In the Drosophila brain, AP1+ senescent glia arise from neuronal mitochondrial dysfunction, promote lipid accumulation in non-senescent glia, and increase senescence markers. While suppressing AP1+ activity extends lifespan, it also exacerbates oxidative damage in the brain, leaving neuronal mitochondrial dysfunction unresolved, possibly due to reduced lipid accumulation.

Highlighted technique: In recent years, many studies have reported a link between senescence and Lipid droplets (LD). LDs are detected with BODIPY 493/503 as used in this paper. We also offer Lipi-Dye, a dye specifically designed for LD detection with high intracellular retention. With its range of color variations, it can be a valuable tool for researchers studying LD.

Previous Science Note

Chronic inflammation drives immune decline and age-related diseases, creating a vicious cycle with senescence. This week, we  introduce a review of the link between senescence and inflammation, along with recent discoveries.

Cellular senescence and inflammation are linked, as senescent cells secrete pro-inflammatory factors that perpetuate chronic inflammation and tissue dysfunction. Chronic inflammation drives immune decline and age-related diseases, creating a vicious cycle with senescence. In this note, we introduce a review article of the link between senescence and inflammation, along with recent findings on anti-IL-17 and anti-IL-11 treatment for anti-senescence. Anti-IL-17 drugs are already approved, and anti-IL-11 therapy is in trials for fibrotic lung disease, suggesting that these treatments may soon help delay aging.

 Review Article   Inflammation and aging: signaling pathways and intervention therapies
Click here for the original article: Xia Li, et. al., Signal Transduct Target Ther., 2023.

Inhibition of IL-11 signalling extends mammalian healthspan and lifespan
Click here for the original article: Anissa A. Widjaja, et. al., Nature, 2024.

Point of Interest
- Chronic inflammation drives aging by promoting cellular senescence, immune dysfunction, and organ damage leading to age-related diseases.

- Inflammaging creates a vicious cycle of inflammation and senescence, suggesting the elimination of inflammation as a potential anti-aging strategy.

- The paper reviews inflammaging, aging models, single-cell technologies, and anti-aging strategies to combat disease and improve quality of life.

Point of Interest
- IL-11 promotes aging by regulating the ERK-AMPK-mTORC1 axis, leading to age-related diseases and reduced lifespan.

- Genetic deletion of IL-11 or use of anti-IL-11 improves metabolism, reduces frailty and extends lifespan by over 20% in mice.

- Anti-IL-11 therapy, currently in clinical trials for fibroinflammatory diseases, may extend health and lifespan in humans with promising safety.
 

Point of Interest
- Skin aging involves structural and functional changes driven by a pro-inflammatory microenvironment and stem cell-intrinsic alterations.

- Single-cell RNA sequencing reveals increased IL-17 signaling in aged skin, which drives inflammation and impairs homeostasis.

- Blocking IL-17 signaling reduces skin inflammation and delays age-related features, suggesting a potential anti-aging skin therapy.

Metabolic shift to glycolysis in senescenct cells

NAD(+) levels decline during the aging process, causing defects in nuclear and mitochondrial functions and resulting in many age-associated pathologies*. Here, we try to redemonstrate this phenomenon in the doxorubicin (DOX)-induced cellular senescence model with a comprehensive analysis of our products.

*S. Imai, et al., Trends Cell Biol, 2014, 24, 464-471

Products in Use
① DNA Damage Detection Kit - γH2AX
② Cellular Senescence Detection Kit - SPiDER-βGal
③ NAD/NADH Assay Kit-WST
④ JC-1 MitoMP Detection Kit
⑤ Glycolysis/OXPHOS Assay Kit、Lactate Assay Kit-WST

Multiple staining with oxidative stress-related markers using Doxorubicin-induced senescent cells（flow cytometry)

Using A549 cells induced to senescence by doxorubicin (DOX) and normal cells (CTRL), changes in oxidative stress-related markers in senescent cells were analyzed by flow cytometry with multiple staining. SA-βGal as a senescence marker was detected by Cellular Senescence Detection Kit - SPiDER Blue, total ROS as an oxidative stress marker was detected by ROS Assay Kit - Photo-oxidation Resistant DCFH-DA-, and γH2AX as a DNA damage marker was detected by DNA Damage Detection Kit - γH2AX-Red. As a result, total ROS and γH2AX were increased in SA-βGal-positive senescent cells, and the increase in oxidative stress-related markers associated with cellular senescence could be detected by multiple staining.


  Flow cytometry：SONY SA3800
　 SPiDER Blue: PacificBlue 　
    ROS Assay Kit: FITC
    γH2AX - Red: Cy3

＜Experimental Procedure>
 *Cellular senescence was induced in A549 cells by DOX (0.2 μM DOX for 3 days → normal medium for 3 days)
 The detail procedure for this experiment, please refer to the product page: SPiDER Blue.

Recent research on cellular senescence reveals that senescent cells contribute to tumor promotion. Here are some papers that highlight how senescent cells create a pro-tumorigenic microenvironment by enhancing inflammation, angiogenesis, and immune evasion, offering therapeutic opportunities.

Cellular senescence is a state of stable cell cycle arrest that prevents the proliferation of damaged or aged cells and acts as a tumor suppressor mechanism. However, senescent cells can secrete a pro-inflammatory and tissue-altering mix of factors that can paradoxically promote cancer progression. Senescent factors contribute to a pro-tumorigenic microenvironment by enhancing inflammation, angiogenesis, and immune evasion. Understanding the dual role of senescence in cancer highlights the complexity of its contribution to tumor suppression and progression and offers potential therapeutic opportunities.

Hematopoietic aging promotes cancer by fueling IL-1⍺–driven emergency myelopoiesis
Click here for the original article: Matthew D. Park, et. al., Science, 2024.

Age-dependent loss of HAPLN1 erodes vascular integrity via indirect upregulation of endothelial ICAM1 in melanoma
Click here for the original article: Gloria E. Marino-Bravante, et. al., Nature Aging, 2024.

Point of Interest
- The aging immune system drives lung cancer progression by increasing myelopoiesis and IL-1⍺ production, which is associated with poorer survival.

- DNMT3A (DNA methyltransferase 3A) downregulation with age increases IL-1⍺ production, driving the recruitment of immunosuppressive myeloid cells and tumor progression.

- Blocking the IL-1 receptor slows lung cancer progression in aged mice, highlighting potential therapeutic strategies for age-related cancers.

Point of Interest
- Age-dependent loss of the extracellular matrix (ECM) protein HAPLN1 compromises vascular integrity in melanoma by indirectly increasing endothelial ICAM1 expression. 

- ICAM1 leads to VE-cadherin internalization, which increases vascular permeability and melanoma progression.

- Blocking ICAM1 reduces tumor size and metastasis in aged mice, highlighting the impact of aging and ECM changes on tumor progression.

Point of Interest
- Aged fibroblasts secrete growth/differentiation factor 15 (GDF-15), which promotes tumor growth and activates AKT signaling in the aged pancreatic cancer microenvironment.

- Treatment of young mice with GDF-15 enhances tumor growth, whereas GDF-15 knockout in aged mice reduces tumor growth.

- AKT inhibition is effective in aged but not young microenvironments, providing a targeted therapy for age-related pancreatic cancer.

Metabolic shift to glycolysis in senescenct cells

NAD(+) levels decline during the aging process, causing defects in nuclear and mitochondrial functions and resulting in many age-associated pathologies*. Here, we try to redemonstrate this phenomenon in the doxorubicin (DOX)-induced cellular senescence model with a comprehensive analysis of our products.

*S. Imai, et al., Trends Cell Biol, 2014, 24, 464-471

Products in Use
① DNA Damage Detection Kit - γH2AX
② Cellular Senescence Detection Kit - SPiDER-βGal
③ NAD/NADH Assay Kit-WST
④ JC-1 MitoMP Detection Kit
⑤ Glycolysis/OXPHOS Assay Kit、Lactate Assay Kit-WST

Multiple staining with oxidative stress-related markers using Doxorubicin-induced senescent cells（flow cytometry)

Using A549 cells induced to senescence by doxorubicin (DOX) and normal cells (CTRL), changes in oxidative stress-related markers in senescent cells were analyzed by flow cytometry with multiple staining. SA-βGal as a senescence marker was detected by Cellular Senescence Detection Kit - SPiDER Blue, total ROS as an oxidative stress marker was detected by ROS Assay Kit - Photo-oxidation Resistant DCFH-DA-, and γH2AX as a DNA damage marker was detected by DNA Damage Detection Kit - γH2AX-Red. As a result, total ROS and γH2AX were increased in SA-βGal-positive senescent cells, and the increase in oxidative stress-related markers associated with cellular senescence could be detected by multiple staining.


  Flow cytometry：SONY SA3800
　 SPiDER Blue: PacificBlue 　
    ROS Assay Kit: FITC
    γH2AX - Red: Cy3

＜Experimental Procedure>
 *Cellular senescence was induced in A549 cells by DOX (0.2 μM DOX for 3 days → normal medium for 3 days)
 The detail procedure for this experiment, please refer to the product page: SPiDER Blue.

Recent research on cellular senescence shows that senescent cells propagate the effects of aging and contribute to chronic inflammation and diseases such as neurodegeneration. Here are studies that explore the systemic effects of senescence and therapeutic strategies that target senescent cells and their secreted factors.

Senescent cells are dysfunctional cells that accumulate in organs and tissues with age and secrete bioactive factors, collectively known as the senescence-associated secretory phenotype (SASP). These factors influence the local microenvironment and can propagate senescence to nearby and distant cells, leading to systemic effects. Systemic senescence contributes to age-related tissue dysfunction, chronic inflammation and the progression of diseases such as cancer, cardiovascular disease and neurodegeneration. Targeting senescent cells or their secreted factors offers a promising therapeutic strategy to attenuate systemic aging and related pathologies.

Hepatocellular senescence induces multi-organ senescence and dysfunction via TGFβ
Click here for the original article: Christos Kiourtis, et. al., Nature Cell Biology, 2024.

Aged bone marrow macrophages drive systemic aging and age-related dysfunction via extracellular vesicle-mediated induction of paracrine senescence
Click here for the original article: Jing Hou, et. al., Nature Aging, 2024.

Point of Interest
- Cellular senescence extends beyond aging to embryonic development, wound healing, and multi-organ dysfunction in liver disease.

- Hepatocyte senescence predicts the outcome of acute liver failure, including organ failure, driven by the systemic effects of the TGFβ signaling pathway.

- Blocking TGFβ inhibits the spread of senescence and prevents liver-induced kidney dysfunction, highlighting the role of senescence in multi-organ dysfunction.

Point of Interest
- Aged bone marrow macrophages (BMMs) spread senescence to tissues via extracellular vesicles and drive age-related dysfunction through PPARα-regulated pathways.

- The PPARα agonist fenofibrate restores tissue homeostasis in aged mice and reduces the risk of chronic diseases, suggesting a potential to extend human lifespan.

- BMMs are key to senescence propagation, linking aging dysfunction to a treatable mechanism with therapeutic implications.

Point of Interest
- Transplantation of senescent fibroblasts into young mice induces senescence in both proximal and distal tissues, causing widespread aging effects.

- The senescent fibroblasts in the skin increase mobility and musculoskeletal impairment, demonstrating systemic effects of skin-resident senescence.

- Skin senescence correlates with hippocampal dysfunction, linking local senescence to brain dysfunction and age-related cognitive decline.

Co-staining with Lipid droplet and SA-β-Gal in fixed cells

NAD(+) levels decline during the aging process, causing defects in nuclear and mitochondrial functions and resulting in many age-associated pathologies*. Here, we try to redemonstrate this phenomenon in the doxorubicin (DOX)-induced cellular senescence model with a comprehensive analysis of our products.

*S. Imai, et al., Trends Cell Biol, 2014, 24, 464-471

Products in Use
① DNA Damage Detection Kit - γH2AX
② Cellular Senescence Detection Kit - SPiDER-βGal
③ NAD/NADH Assay Kit-WST
④ JC-1 MitoMP Detection Kit
⑤ Glycolysis/OXPHOS Assay Kit、Lactate Assay Kit-WST

Multiple staining with oxidative stress-related markers using Doxorubicin-induced senescent cells（flow cytometry)

Using A549 cells induced to senescence by doxorubicin (DOX) and normal cells (CTRL), changes in oxidative stress-related markers in senescent cells were analyzed by flow cytometry with multiple staining. SA-βGal as a senescence marker was detected by Cellular Senescence Detection Kit - SPiDER Blue, total ROS as an oxidative stress marker was detected by ROS Assay Kit - Photo-oxidation Resistant DCFH-DA-, and γH2AX as a DNA damage marker was detected by DNA Damage Detection Kit - γH2AX-Red. As a result, total ROS and γH2AX were increased in SA-βGal-positive senescent cells, and the increase in oxidative stress-related markers associated with cellular senescence could be detected by multiple staining.


  Flow cytometry：SONY SA3800
　 SPiDER Blue: PacificBlue 　
    ROS Assay Kit: FITC
    γH2AX - Red: Cy3

＜Experimental Procedure>
 *Cellular senescence was induced in A549 cells by DOX (0.2 μM DOX for 3 days → normal medium for 3 days)
 The detail procedure for this experiment, please refer to the product page: SPiDER Blue.

Recent cancer research has focused on intratumoral senescent cancer cells and it is becoming clear that these cells contribute to immune evasion of cancer cells and create a cancer-promoting environment.  Here are some of the papers that have identified senescent cells or senescent cells with specific phenotypes in cancer treatment and have shown that removing these cells is important for cancer treatment.

Senescent cells are damaged or stressed cells that permanently stop dividing but do not undergo cell death. In the context of cancer, senescent cells can accumulate in tumors, particularly after treatments such as chemotherapy, contributing to an immunosuppressive environment. This environment can promote tumor growth, immune evasion and resistance to therapy. However, the selective targeting and elimination of senescent cells, known as senolytic therapy, is emerging as a promising strategy to improve cancer treatment by reducing tumor progression and improving patient outcomes.

The efficacy of chemotherapy is limited by intratumoral senescent cells expressing PD-L2
Click here for the original article: Selim Chaib, et. al., Nature Cancer, 2024.

Cellular senescence in malignant cells promotes tumor progression in mouse and patient Glioblastoma
Click here for the original article: Rana Salam, et. al., Nature Communications, 2023.

Point of Interest
- Chemotherapy often generates senescent cancer cells, which have increased PDL-2 expression, aiding immune evasion and tumor growth.

- PD-L2 is not necessary for senescence but crucial for immune evasion by senescent cells, promoting tumor persistence after chemotherapy.

- Antibody-mediated PD-L2 blockade synergizes with chemotherapy, suggesting a potential therapeutic strategy targeting senescent cancer cells.

Point of Interest
- Senescent cells contribute to glioblastoma (GBM) progression and their elimination may improve treatment outcomes.

- Removal of p16Ink4a-expressing senescent cells from GBM tumors improves survival in mice and alters the tumour environment.

- Senolytic drugs that target senescent cells may be a promising adjuvant therapy for GBM patients, improving survival.
 

Point of Interest
- Immunotherapy resistance is associated with reduced CD8 T cell activity in tumors.

- Elimination of senescent cells restores CD8 T cell proliferation and reduces immunotherapy resistance.

- Anti-senescent cell drugs prior to immune checkpoint inhibitors may enhance the effectiveness of cancer immunotherapy.

Co-staining with Lipid droplet and SA-β-Gal in fixed cells

NAD(+) levels decline during the aging process, causing defects in nuclear and mitochondrial functions and resulting in many age-associated pathologies*. Here, we try to redemonstrate this phenomenon in the doxorubicin (DOX)-induced cellular senescence model with a comprehensive analysis of our products.

*S. Imai, et al., Trends Cell Biol, 2014, 24, 464-471

Products in Use
① DNA Damage Detection Kit - γH2AX
② Cellular Senescence Detection Kit - SPiDER-βGal
③ NAD/NADH Assay Kit-WST
④ JC-1 MitoMP Detection Kit
⑤ Glycolysis/OXPHOS Assay Kit、Lactate Assay Kit-WST

Multiple staining with oxidative stress-related markers using Doxorubicin-induced senescent cells（flow cytometry)

Using A549 cells induced to senescence by doxorubicin (DOX) and normal cells (CTRL), changes in oxidative stress-related markers in senescent cells were analyzed by flow cytometry with multiple staining. SA-βGal as a senescence marker was detected by Cellular Senescence Detection Kit - SPiDER Blue, total ROS as an oxidative stress marker was detected by ROS Assay Kit - Photo-oxidation Resistant DCFH-DA-, and γH2AX as a DNA damage marker was detected by DNA Damage Detection Kit - γH2AX-Red. As a result, total ROS and γH2AX were increased in SA-βGal-positive senescent cells, and the increase in oxidative stress-related markers associated with cellular senescence could be detected by multiple staining.


  Flow cytometry：SONY SA3800
　 SPiDER Blue: PacificBlue 　
    ROS Assay Kit: FITC
    γH2AX - Red: Cy3

＜Experimental Procedure>
 *Cellular senescence was induced in A549 cells by DOX (0.2 μM DOX for 3 days → normal medium for 3 days)
 The detail procedure for this experiment, please refer to the product page: SPiDER Blue.

Recent research on senescence is revealing that senescence is related to lipid droplet and lysosomal dysfunction, which impair cell function. Here are some of the papers showing that these processes leads to neurodegenerative diseases.

Cellular senescence, a state of irreversible growth arrest, is closely linked to neurodegeneration through the accumulation of damaged cells in the nervous system. Lipid droplets, which store excess lipids, can accumulate in aging or stressed cells, contributing to cellular dysfunction and exacerbating neurodegenerative processes. Lysosomal dysfunction plays a central role in both lipid accumulation and the removal of cellular waste, and its impairment leads to the accumulation of toxic substances that further drive neurodegeneration. Together, these mechanisms create a cycle of cellular damage that accelerates the progression of neurodegenerative diseases such as Alzheimer's and Parkinson's.

Point of Interest
- Parkinson's disease (PD) is associated with the loss of dopaminergic neurons, with genetic and environmental factors contributing to its progression.

- Mutations in the lysosomal enzyme β-glucocerebrosidase cause lipid accumulation that drives cellular senescence in dopaminergic neurons in PD.

- Lipid droplet aggregation and lysosomal dysfunction may trigger cellular senescence leading to neurodegeneration in PD.

Point of Interest
- Senescent glia in aging Drosophila brains originate from neuronal mitochondrial dysfunction and express AP1, a senescence-associated transcription factor.

- AP1⁺ senescent glia cause lipid accumulation in non-senescent glia and increase senescence markers.

- Targeting AP1 in senescent glia extends lifespan, but increases oxidative damage in the brain and neuronal mitochondrial function remains poor.

Point of Interest
- Alzheimer's disease risk genes influence macrophage and microglial responses in lipid-rich tissues such as the brain.

- DLAMs are macrophage subpopulations with similar transcriptional activation states found in aging brains and other diseased lipid-rich tissues.

- Targeting BHLHE40/41, transcriptional regulators of DALM, may improve cholesterol clearance and lysosomal function in Alzheimer's disease therapies.

Co-staining with Lipid droplet and SA-β-Gal in fixed cells

*Cellular senescence was induced in A549 cells by DOX (0.2 μM DOX for 3 days → normal medium for 3 days)

1. A549 (2 x 10⁴) cells were seeded onto µ-slide 8 well plates (ibidi) and cultured overnight in a 37°C CO₂ incubator. 
2. The supernatant was removed, washed once with PBS, and fixed in 4% paraformaldehyde (PFA)/PBS solution for 30 minutes at room temperature. 
3. The supernatant was removed and the cells were washed once with PBS. 
4. 15 µM SPiDER Blue + 0.1 µM Lipi-Deep Red prepared in Assay buffer was added and incubated at 37°C for 30 min.
5. The supernatant was removed, washed once with PBS, and 200 µl of PBS was added and observed under a confocal laser microscope (60x magnification).

Imaging analysis of lipid droplet accumulation in senescent cells was performed using normal A549 cells (CTRL)  and cells induced senescence by Doxorubicin treatment (DOX). SA-β-Gal was detected as a senescence marker with Cellular Senescence Detection Kit - SPiDER Blue, and lipid droplets were detected with Lipi-Deep Red. As a result, the signal of Lipi-Deep Red was increased in SA-β-Gal-positive senescent cells.

[Detection conditions]

SPiDE Blue: 405 nm (Ex), 400–550 nm (Em), 1.0%, 600V
Lipi-Deep Red: 633 nm (Ex), 650–700 nm (Em), 1.0%, 650V

Multiple staining with oxidative stress-related markers using Doxorubicin-induced senescent cells（flow cytometry)

Using A549 cells induced to senescence by doxorubicin (DOX) and normal cells (CTRL), changes in oxidative stress-related markers in senescent cells were analyzed by flow cytometry with multiple staining. SA-βGal as a senescence marker was detected by Cellular Senescence Detection Kit - SPiDER Blue, total ROS as an oxidative stress marker was detected by ROS Assay Kit - Photo-oxidation Resistant DCFH-DA-, and γH2AX as a DNA damage marker was detected by DNA Damage Detection Kit - γH2AX-Red. As a result, total ROS and γH2AX were increased in SA-βGal-positive senescent cells, and the increase in oxidative stress-related markers associated with cellular senescence could be detected by multiple staining.


  Flow cytometry：SONY SA3800
　 SPiDER Blue: PacificBlue 　
    ROS Assay Kit: FITC
    γH2AX - Red: Cy3

＜Experimental Procedure>
 *Cellular senescence was induced in A549 cells by DOX (0.2 μM DOX for 3 days → normal medium for 3 days)
 The detail procedure for this experiment, please refer to the product page: SPiDER Blue.

Anti-senescence strategies often target metabolic pathways to delay or reverse cellular aging processes. Key pathways such as the pentose phosphate pathway (PPP) and mitochondrial metabolism are modulated to reduce oxidative stress and increase cellular resilience. By optimizing these pathways, anti-senescence approaches aim to maintain energy production, prevent accumulation of damage, and preserve stem cell function. Ultimately, these interventions may improve tissue homeostasis and mitigate age-related decline.

Point of Interest
- A switch that results in the accumulation of glycerol-3-phosphate (G3P) and phosphoethanolamine (pEtN) links lipid metabolism to the senescence gene expression program in human fibroblasts.

- p53-dependent glycerol kinase activation and post-translational modifications drive this metabolic switch, promoting triglyceride accumulation and senescence.

- Scavenging G3P and pEtN reduces their accumulation, acting senomorphically and potentially providing a therapeutic approach to target senescence.

Point of Interest
- The pluripotency transcription factor NANOG rejuvenates senescent mitochondria by restoring the key proline biosynthetic enzymes PYCR1 and PYCR2, thereby improving cellular metabolism.

- Proline supplementation induces mitophagy, alleviates mitochondrial respiratory impairment and improves mitochondrial clearance in senescent cells.

- Proline treatment mitigates the hallmarks of aging, including DNA damage, inflammation, and impaired differentiation, by restoring mitochondrial function.

Point of Interest
- ACLY, critical for acetyl-CoA synthesis, drives the pro-inflammatory SASP in senescent cells independent of growth arrest.

- ACLY-dependent acetyl-CoA activates SASP gene enhancers, increasing BRD4 recruitment and promoting SASP activation.

- Chemical inhibition of the ACLY-BRD4 axis suppresses SASP in aged mice, providing a potential target for promoting healthy aging.

Metabolic shift to glycolysis in senescenct cells

NAD(+) levels decline during the aging process, causing defects in nuclear and mitochondrial functions and resulting in many age-associated pathologies*. Here, we try to redemonstrate this phenomenon in the doxorubicin (DOX)-induced cellular senescence model with a comprehensive analysis of our products.

*S. Imai, et al., Trends Cell Biol, 2014, 24, 464-471

Products in Use
① DNA Damage Detection Kit - γH2AX
② Cellular Senescence Detection Kit - SPiDER-βGal
③ NAD/NADH Assay Kit-WST
④ JC-1 MitoMP Detection Kit
⑤ Glycolysis/OXPHOS Assay Kit、Lactate Assay Kit-WST

Anti-senescence study aims to restore youthful function to aging tissues and organs, often focusing on cellular repair and regeneration. Extracellular vesicles (EVs), including exosomes and microvesicles, play a critical role in cell-to-cell communication by transporting bioactive molecules such as proteins, lipids and RNA. Recent studies suggest that EVs derived from young or healthy cells can have anti-senescence effects on aged cells and tissues, promoting regeneration and repair. These EVs have shown potential to reverse age-related damage and improve tissue function, making them a promising tool in regenerative medicine and anti-aging therapies.

Small extracellular vesicles from young plasma reverse age-related functional declines by improving mitochondrial energy metabolism
Click here for the original article: Xiaorui Chen, et. al., Nature Aging, 2024.

Point of Interest
- Young sEVs rejuvenate aged mice tissues and extend lifespan.

- Proteomic analysis shows young sEVs enhance metabolism and mitochondrial function in aged tissues.

- Young sEVs stimulate PGC-1α, reversing age-related dysfunction.

Point of Interest
- Intravenous injection of human urine-derived stem cell EVs (USC-EVs) inhibit cellular senescence, improving cognitive and physical functions in mice.

- USC-EVs' anti-aging effects are consistent regardless of donor age, gender, or health.

- Plasminogen activator urokinase (PLAU) and tissue inhibitor of metalloproteinases 1 (TIMP1) in USC-EVs contribute to the anti-senescent effects of USC-EVs.

- Purified EVs from young blood show rejuvenating effects; EV-depleted serum increases senescence.

- The treatment also favorably modulates glucose metabolism and anti-senescence pathways, ultimately extending lifespan.

Metabolic shift to glycolysis in senescenct cells

NAD(+) levels decline during the aging process, causing defects in nuclear and mitochondrial functions and resulting in many age-associated pathologies*. Here, we try to redemonstrate this phenomenon in the doxorubicin (DOX)-induced cellular senescence model with a comprehensive analysis of our products.

*S. Imai, et al., Trends Cell Biol, 2014, 24, 464-471

Products in Use
① DNA Damage Detection Kit - γH2AX
② Cellular Senescence Detection Kit - SPiDER-βGal
③ NAD/NADH Assay Kit-WST
④ JC-1 MitoMP Detection Kit
⑤ Glycolysis/OXPHOS Assay Kit、Lactate Assay Kit-WST

Senescence is a cellular process that results in the cessation of cell division, often serving as a protective mechanism against the proliferation of damaged cells, including potential cancer cells. This process is intricately regulated by numerous factors including, but not limited to, tumor suppressor genes, DNA damage response (DDR) pathways, and various signaling molecules. In addition, the senescence-associated secretory phenotype (SASP), consisting of cytokines, growth factors, and proteases, is regulated by NF-κB and other transcription factors that influence the tissue microenvironment and impact aging and disease processes.

HKDC1, a target of TFEB, is essential to maintain both mitochondrial and lysosomal homeostasis, preventing cellular senescence
Click here for the original article: Mengying Cui, et. al., PNAS, 2023.

Point of Interest
- HKDC1, a protein involved in glycolysis, is a direct target of the transcription factor TFEB and is essential for maintaining both mitochondrial and lysosomal function.

- This activity helps avert cellular senescence, playing a vital role in maintaining cellular homeostasis.

- Beyond its role in glycolysis, HKDC1 contributes to mitophagy and lysosomal repair processes independently.

- The absence of HKDC1 may result in cellular senescence and the buildup of damaged organelles.

Point of Interest
- Vascular and hemolytic injury trigger iron accumulation, which causes senescence and promotes fibrosis.

- Senescent cells persistently accumulate iron, even after the increase in extracellular iron has subsided.

- Cells exposed to various types of senescence-inducing insults accumulate abundant ferritin-bound iron, mostly within lysosomes.
- The high levels of labile iron fuel the generation of reactive oxygen species and the SASP.  

- STK38 and GABARAPs are key regulators of this process.

- STK38 is required for lysosomal recruitment of VPS4 and GABARAPs are involved in ESCRT assembly.

- Depletion of these regulators leads to accelerated cellular senescence and shortened lifespan.

Analysis of Lysosomal Mass and pH change in Senescence-induced Cells

Purpose: To investigate changes in lysosomal mass and pH in A549 cells induced to senescence by treatment with Doxorubicin (DOX).

Methods: Senescence-associated acidic β-galactosidase (SA-βGal) activity was detected using Cellular Senescence Detection Kit - SPiDER-βGal. Lysosomal mass was detected using LysoPrime Deep Red, and pH was detected using pHLys Red. Fluorescence imaging was used to observe changes in lysosomal mass and pH in senescent cells compared to non-senescent cells. The normalized fluorescence intensity of lysosomal mass and pH was also measured by a plate reader.

Results: Our findings indicate that senescence induced by DOX resulted in an increase in lysosomal mass and acidification of pH compared to non-senescent cells. The obtained results are consistent with previous reports* that demonstrated enhanced lysosomal activity in senescent cells induced by the CDK4/6 inhibitor, palbociclib. The fluorescence imaging and plate reader data both support these findings.

* Miguel Rovira, et. al., Aging Cell (2022)

＜Experimental Conditions for Microscopy＞
SA-βGal(Green):Ex = 488 nm, Em = 490 – 550 nm
Lysosomal pH (Red):Ex = 561 nm, Em = 560 – 620 nm
Lysosomal mass (Deep Red):Ex = 633 nm, Em = 640 – 700 nm

＜Experimental Conditions for Plate Reader＞
SA-βGal: Ex = 525 – 535 nm, Em = 550 – 570 nm
Lysosomal pH: Ex = 555 – 565 nm, Em = 590 – 610 nm
Lysosomal mass: Ex = 645 – 655 nm, Em = 690 – 710 nm

<Products in Use>
- Cellular Senescence Detection Kit
- Lysosomal pH and mass detection Kit
   > More about Lysosomal Function Analysis

Scientists have discovered that, in the absence of resident stem cells, senescent cells can instruct neighboring somatic cells to reprogram. This reprogramming enables them to become stem cells, driving whole-body regeneration in the cnidarian Hydractinia symbiolongicarpus.

Senescence-induced cellular reprogramming drives cnidarian whole-body regeneration
Click here for the original article: Miguel Salinas-Saavedra, et. al., Cell Reports, 2023.

Point of Interest
- Amputation injury induces senescence in a small number of specific head cells in Hydractinia
- Senescent cells persist in the tissue for several hours before being expelled
- Signals emitted by senescent cells induce the reprogramming of neighboring cells
- These reprogrammed cells proliferate and drive the process of whole-body regeneration

Irreversible cell cycle arrest is one of the phenomena that characterize cellular senescence. p16, p21, p53, and pRB (phosphorylated retinoblastoma protein) are known as representative protein markers. The activation/upregulation of these proteins are used as indicators of cellular senescence. These marker proteins are known to be tumor suppressors and regulate the cell cycle mainly through two pathways (p16Ink4a-RB and p53-p21CIP1).

Doxorubicin (DOX) is known as an anticancer drug that acts in the G2/M phase of the cell cycle to arrest cell proliferation and induce cellular senescence (see the upper left figure).  The figure on the left shows the results of an experiment in which DOX was added to A549 cells. As a result, changes in SA-ß-Gal expression, cell cycle progression, and mitochondrial membrane potential were observed.

- Cell Cycle Assay Solution Blue / Deep Red
- JC-1 MitoMP Detection Kit
- Cellular Senescence Detection Kit

Scientists have discovered that obesity and dyslipidemia are linked to dysregulated DNA hydroxymethylation in apoptosis- and senescence-related genes in both swine and human MSCs. This dysregulation might impact cell vitality and regenerative capabilities. Vitamin C could mediate the reprogramming of this altered epigenomic profile, offering a potential approach to enhance the success of autologous MSC transplantation in obese patients.

Obesity and dyslipidemia are associated with partially reversible modifications to DNA hydroxymethylation of apoptosis- and senescence-related genes in swine adipose-derived mesenchymal stem/stromal cells
Click here for the original article: Logan M. Glasstetter, et. al., Stem Cell Research & Therapy, 2023.

Point of Interest
- hMeDIP-seq identified 467 hyper- and 591 hypo-hydroxymethylated loci in swine Obese- versus Lean-MSCs.
- Combined hMeDIP-seq/mRNA-seq showed dysregulated genes related to apoptosis, cell proliferation, and senescence.  
- These changes were linked to increased senescence in MSCs and were partly reversed with Vitamin C.  
- Similar pathways were observed in human obese-MSCs.

Metabolic shift to glycolysis in senescenct cells

NAD(+) levels decline during the aging process, causing defects in nuclear and mitochondrial functions and resulting in many age-associated pathologies*. Here, we try to redemonstrate this phenomenon in the doxorubicin (DOX)-induced cellular senescence model with a comprehensive analysis of our products.

*S. Imai, et al., Trends Cell Biol, 2014, 24, 464-471

Products in Use
① DNA Damage Detection Kit - γH2AX
② Cellular Senescence Detection Kit - SPiDER-βGal
③ NAD/NADH Assay Kit-WST
④ JC-1 MitoMP Detection Kit
⑤ Glycolysis/OXPHOS Assay Kit、Lactate Assay Kit-WST

Through single-cell RNA sequencing (scRNA-seq), they identified an indispensable transcription factor, CBFb that ensures AM self-renewal. The study concludes that AMs in aged hosts exhibit signs of senescence, possibly due to reduced activity of the CBFb transcription factor.

Single cell RNA sequencing unravels mechanisms underlying senescence-like phenotypes of alveolar macrophages
Click here for the original article: Yue Wu, et. al., iScience, 2023.

Point of Interest
- Damaged and aged alveolar macrophages (AMs) showed reduced embryonic stem-cell-like features and compromised DNA repair abilities, respectively.
- These deficiencies were associated with a decrease in self-renewal capabilities and cellular senescence.
- AMs in aged hosts exhibit signs of senescence, possibly due to reduced activity of the CBFb transcription factor.

Purpose: To investigate changes in lysosomal mass and pH in A549 cells induced to senescence by treatment with Doxorubicin (DOX).

Methods: Senescence-associated acidic β-galactosidase (SA-βGal) activity was detected using Cellular Senescence Detection Kit - SPiDER-βGal. Lysosomal mass was detected using LysoPrime Deep Red, and pH was detected using pHLys Red. Fluorescence imaging was used to observe changes in lysosomal mass and pH in senescent cells compared to non-senescent cells. The normalized fluorescence intensity of lysosomal mass and pH was also measured by a plate reader.

Results: Our findings indicate that senescence induced by DOX resulted in an increase in lysosomal mass and acidification of pH compared to non-senescent cells. The obtained results are consistent with previous reports* that demonstrated enhanced lysosomal activity in senescent cells induced by the CDK4/6 inhibitor, palbociclib. The fluorescence imaging and plate reader data both support these findings.

* Miguel Rovira, et. al., Aging Cell (2022)

＜Experimental Conditions for Microscopy＞
SA-βGal(Green):Ex = 488 nm, Em = 490 – 550 nm
Lysosomal pH (Red):Ex = 561 nm, Em = 560 – 620 nm
Lysosomal mass (Deep Red):Ex = 633 nm, Em = 640 – 700 nm

＜Experimental Conditions for Plate Reader＞
SA-βGal: Ex = 525 – 535 nm, Em = 550 – 570 nm
Lysosomal pH: Ex = 555 – 565 nm, Em = 590 – 610 nm
Lysosomal mass: Ex = 645 – 655 nm, Em = 690 – 710 nm

<Products in Use>
- Cellular Senescence Detection Kit
- Lysosomal pH and mass detection Kit

  More about Lysosomal Function Analysis

Scientists have unveiled that senescence is irreversible and that commitment to and maintenance of senescence is mediated by irreversible MYC degradation.  Thus, irreversible cell cycle exit associated with senescence is mediated by constitutive MYC degradation, but bypassing this degradation may allow tumor cells to escape during cancer initiation.

Irreversible cell cycle exit associated with senescence is mediated by constitutive MYC degradation
Click here for the original article: Marwa M. Afifi, et. al., Cell Reports, 2023.

Point of Interest
- The likelihood of cells entering an irreversible state of senescence rises with time.
- Permanent cessation of the cell cycle is facilitated by the loss of MYC.
- The expression of non-degradable MYC enables senescent cells to resume division.
- Oral premalignant lesions evade oncogene-induced senescence through MYC overexpression.

Metabolic shift to glycolysis in senescenct cells

NAD(+) levels decline during the aging process, causing defects in nuclear and mitochondrial functions and resulting in many age-associated pathologies*. Here, we try to redemonstrate this phenomenon in the doxorubicin (DOX)-induced cellular senescence model with a comprehensive analysis of our products.

*S. Imai, et al., Trends Cell Biol, 2014, 24, 464-471

Products in Use
① DNA Damage Detection Kit - γH2AX
② Cellular Senescence Detection Kit - SPiDER-βGal
③ NAD/NADH Assay Kit-WST
④ JC-1 MitoMP Detection Kit
⑤ Glycolysis/OXPHOS Assay Kit、Lactate Assay Kit-WST

In recent years, the discovery of several novel senescence-related immune and neurological responses has attracted significant attention. The stress associated with mitochondrial protein folding is a driver of neural stem cell aging and suggests approaches to ameliorate aging-associated cognitive decline. In other breakthroughs, targeting bone marrow-derived senescent immune cells offers an avenue for improving brown adipose tissue aging and related metabolic disorders. Research also reveals the protective role of microglial autophagy in regulating the homeostasis of amyloid plaques and in preventing senescence; thus, the removal of senescent microglia emerges as a promising therapeutic strategy.

The mitochondrial unfolded protein response regulates hippocampal neural stem cell aging
Click here for the original article: Chih-Ling Wang , et. al., Cell Metabolism, 2023.

Senescent immune cells accumulation promotes brown adipose tissue dysfunction during aging
Click here for the original article: Xu Feng, et. al., Nature Communications, 2023.

Autophagy enables microglia to engage amyloid plaques and prevents microglial senescence
Click here for the original article: Insup Choi , et. al., Nature Cell Biology, 2023.

Point of Interest
- Mitochondrial protein-folding stress increases in neural stem cells as they age.
- SIRT7 protects neural stem cells by reducing mitochondrial protein-folding stress.
- Overexpression of SIRT7 enhances neurogenesis and improves cognition in aged mice.

Point of Interest
- Senescent immune cells, predominantly T cells and neutrophils, secrete an abundance of S100A8.
- These S100A8-expressing immune cells, in conjunction with adipocytes and sympathetic nerves, lead to a reduction in thermogenic function.
- An S100A8 inhibitor rejuvenates the axon networks within brown adipose tissue and restores thermogenic function in aged male mice.

Point of Interest
- Inhibiting microglial autophagy leads to the disengagement of microglia from amyloid plaques, exacerbating neuropathology in mice with Alzheimer's Disease (AD).
- An autophagy deficiency fosters the development of senescent microglia.
- Pharmacological removal of autophagy-deficient senescent microglia can alleviate neuropathology in mice with AD.

Metabolic shift to glycolysis in senescenct cells

NAD(+) levels decline during the aging process, causing defects in nuclear and mitochondrial functions and resulting in many age-associated pathologies*. Here, we try to redemonstrate this phenomenon in the doxorubicin (DOX)-induced cellular senescence model with a comprehensive analysis of our products.

*S. Imai, et al., Trends Cell Biol, 2014, 24, 464-471

Products in Use
① DNA Damage Detection Kit - γH2AX
② Cellular Senescence Detection Kit - SPiDER-βGal
③ NAD/NADH Assay Kit-WST
④ JC-1 MitoMP Detection Kit
⑤ Glycolysis/OXPHOS Assay Kit、Lactate Assay Kit-WST

Lysosomal control of senescence and inflammation through cholesterol partitioning
Kyeonghwan Roh, et. al., Nature Metabolism (2023)

   Point of Interest
   - Cholesterol is essential for various cellular functions, but its dysregulation can lead to age-related pathologies.
   - Senescent cells have been found to accumulate cholesterol in lysosomes, which helps maintain their SASP.
   - The accumulation of lysosomal cholesterol results in the formation of cholesterol-rich microdomains that sustain mTORC1 activity, supporting SASP.
   - By modulating lysosomal cholesterol partitioning, researchers were able to alter senescence-associated inflammation and osteoarthritis progression in mice, suggesting a potential regulatory role for cholesterol in age-related inflammation.

Purpose: To investigate changes in lysosomal mass and pH in A549 cells induced to senescence by treatment with Doxorubicin (DOX).

Methods: Senescence-associated acidic β-galactosidase (SA-βGal) activity was detected using Cellular Senescence Detection Kit - SPiDER-βGal. Lysosomal mass was detected using LysoPrime Deep Red, and pH was detected using pHLys Red. Fluorescence imaging was used to observe changes in lysosomal mass and pH in senescent cells compared to non-senescent cells. The normalized fluorescence intensity of lysosomal mass and pH was also measured by a plate reader.

Results: Our findings indicate that senescence induced by DOX resulted in an increase in lysosomal mass and acidification of pH compared to non-senescent cells. The obtained results are consistent with previous reports* that demonstrated enhanced lysosomal activity in senescent cells induced by the CDK4/6 inhibitor, palbociclib. The fluorescence imaging and plate reader data both support these findings.

* Miguel Rovira, et. al., Aging Cell (2022)

＜Experimental Conditions for Microscopy＞
SA-βGal(Green):Ex = 488 nm, Em = 490 – 550 nm
Lysosomal pH (Red):Ex = 561 nm, Em = 560 – 620 nm
Lysosomal mass (Deep Red):Ex = 633 nm, Em = 640 – 700 nm

＜Experimental Conditions for Plate Reader＞
SA-βGal: Ex = 525 – 535 nm, Em = 550 – 570 nm
Lysosomal pH: Ex = 555 – 565 nm, Em = 590 – 610 nm
Lysosomal mass: Ex = 645 – 655 nm, Em = 690 – 710 nm

<Products in Use>
- Cellular Senescence Detection Kit
- Lysosomal pH and mass detection Kit

  More about Lysosomal Function Analysis

Suman Rimal, et. al., EMBO Reports, e55548 (2023)

Point of Interest
     - RET is activated in aged Drosophila and Drosophila models of Alzheimer's disease (AD).
     - Inhibition of RET using the small molecule drug CPT or by knocking down the mitochondrial complex I subunit NDUFS3 extends the lifespan in both Drosophila and mice.
     - RET inhibition rescues AD-related disease phenotypes in Drosophila and mouse models.
     - RET activity is also observed in human induced pluripotent stem cell (iPSC) models of AD.            

Metabolic shift to glycolysis in senescenct cells

NAD(+) levels decline during the aging process, causing defects in nuclear and mitochondrial functions and resulting in many age-associated pathologies*. Here, we try to redemonstrate this phenomenon in the doxorubicin (DOX)-induced cellular senescence model with a comprehensive analysis of our products.                        *S. Imai, et al., Trends Cell Biol, 2014, 24, 464-471 Products in Use ① DNA Damage Detection Kit - γH2AX ② Cellular Senescence Detection Kit - SPiDER-βGal ③ NAD/NADH Assay Kit-WST ④ JC-1 MitoMP Detection Kit ⑤ Glycolysis/OXPHOS Assay Kit、Lactate Assay Kit-WST

• - Aged, induced neurons (iNs) endogenously present features of cellular senescence
• - Alzheimer’s neurons senesce more frequently than healthy controls
• - Senescent neurons gain an inflammatory senescence-associated secretory phenotype
• - Senescent neurons can be eliminated with senotherapeutics

• - The authors developed a method for isolating senescent cells from damaged muscles in mice
• - There are different senescent cell types from damaged muscles of young and old mice
• - These senescent cells arrest stem cell proliferation and regeneration by creating an inflamed niche.
• - By reducing senescent cells or neutralizing their inflammatory secretions, tissue regeneration was accreted in young and old mice.

• - A new reporter has been created to detect p16INK4a, a biomarker of cellular senescence.
• - p16INK4a-expressing fibroblasts with certain senescent characteristics appeared near epithelial stem cells in the lung shortly after birth.
• - These p16INK4a+ fibroblasts have a heightened ability to sense tissue inflammation and promote epithelial regeneration.
• - p16INK4a expression is necessary for fibroblasts to enhance epithelial regeneration.

Cellular senescence is a complex biological process that is influenced by several key factors: DNA damage, telomere shortening, oxidative stress, oncogene activation, and so on. These factors collectively contribute to the complex process of cellular senescence, which acts as a double-edged sword - protective in preventing cancer growth, but also contributing to aging and age-related diseases.

Apoptotic stress causes mtDNA release during senescence and drives the SASP
Click here for the original article: Stella Victorelli, et. al., Nature, 2023.

Point of Interest
- Some mitochondrial outer membrane permeabilization (MOMP) requires BAX and BAK macropores.
- These macropores allow the release of mitochondrial DNA (mtDNA) into the cytosol.
- Cytosolic mtDNA in turn activates the cGAS-STING pathway, a key regulator of the SASP.  
- Inhibition of MOMP in vivo reduces inflammatory markers and improves healthspan in aged mice.
 

Metabolic shift to glycolysis in senescenct cells

NAD(+) levels decline during the aging process, causing defects in nuclear and mitochondrial functions and resulting in many age-associated pathologies*. Here, we try to redemonstrate this phenomenon in the doxorubicin (DOX)-induced cellular senescence model with a comprehensive analysis of our products.

*S. Imai, et al., Trends Cell Biol, 2014, 24, 464-471

Products in Use
① DNA Damage Detection Kit - γH2AX
② Cellular Senescence Detection Kit - SPiDER-βGal
③ NAD/NADH Assay Kit-WST
④ JC-1 MitoMP Detection Kit
⑤ Glycolysis/OXPHOS Assay Kit、Lactate Assay Kit-WST