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Correction pp undefined-undefined
Correction for: Iron retardation in lysosomes protects senescent cells from ferroptosis
Relevance score: 6.440936Yujing Feng, Huaiqing Wei, Meng Lyu, Zhiyuan Yu, Jia Chen, Xinxing Lyu, Fengfeng Zhuang
Keywords: iron accumulation, senescent cells, lysosome, ferroptosis, ferritinophagy
Published in Aging on July 31, 2024
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Priority Research Paper Volume 13, Issue 12 pp 15750-15769
Acid ceramidase promotes senescent cell survival
Relevance score: 5.8593297Rachel Munk, Carlos Anerillas, Martina Rossi, Dimitrios Tsitsipatis, Jennifer L. Martindale, Allison B. Herman, Jen-Hao Yang, Jackson A. Roberts, Vijay R. Varma, Poonam R. Pandey, Madhav Thambisetty, Myriam Gorospe, Kotb Abdelmohsen
Keywords: senotherapy, post-transcriptional, SASP, senescent cell metabolism, translational control
Published in Aging on June 8, 2021
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Research Paper Volume 12, Issue 14 pp 15050-15057
Establishing a density-based method to separate proliferating and senescent cells from bone marrow stromal cells
Relevance score: 4.807783Fei Xu, Qiang Zhang, Haitao Wang
Keywords: bone marrow stromal cells, cell transplantation, density-based method, proliferating cells, senescent cells
Published in Aging on July 25, 2020
Isolation and Identification of BMSCs by Phenotypic Characterization and Multipotent Differentiation Potential. (A) Cells were isolated from the femurs and tibias of 3- to 4-week-old mice shown at P0 and P3. Cells are attached at P3. Scale Bar=200μm. (B) Flow cytometric analysis of cell surface markers on isolated BMSCs indicates Scal-1+ CD29+ CD11b- CD45- CD105-. (C) Differentiation capacity of BMSCs: ALP staining of cells cultured in osteogenic induction medium for 7 days (upper-left image); alizarin red staining of cells cultured in osteogenic induction medium for 21 days (upper-right image); oil red O staining of cells cultured in adipogenic induction medium for 7 days (lower-left image); and alcian blue staining of cells cultured in chondrogenic induction medium for 14 days (lower-right image). P0, passage 0; P2, passage 2; P3, passage 3. Scale Bar=200μm.
Density Gradient Separation of proliferating and Senescent Cells. (A) Immunofluorescence of BrdU positive cells is shown in the first panel. The right panel shows that the number of BrdU positive cells is significantly lower in P8 compared to P3. Scale Bar=200μm. (B) BMSCs (P8), a mixture of proliferating and senescent cells, were centrifuged through a density gradient medium (OptiPrep, Sigma-Aldrich) at 800g for 20 minutes. Aliquots (0.5 mL) were taken from the low- and high-density layers. The cells were then incubated in a 48-well plate. (C) SA-β-gal staining of the 2 groups. Low-density cells contained a higher percentage of SA-β-gal positive cells compared to the high-density cells. The right panel shows that the number of SA-β-gal positive cells is significantly lower in high-density cells compared to low-density cells (n=3). BrdU indicates bromodeoxyuridine; DAPI, 4′,6-diamidino-2-phenylindole; P3, passage 3; P8, passage 8; SA-β-gal, senescence-associated β-galactosidase; Paired T-Test **, P<.01. data are represented as mean ± SEM. Scale Bar=200μm.
Differentiation Capacity and TIF Assay of BMSCs After Separation. (A) Co-localization of 53BP1 DNA damage protein (green) and telomeric DNA (red). TIFs are indicated by arrows. Blue, DAPI; Red, Telomere; Orange/Yellow, TIFs. Scale Bar=5μm. (B) The percentage of TIF-positive cells is substantially lower in high-density cells compared to low-density cells (left panel). The protein levels of γH2AX and P21 were induced at low density (LD) using Western blot. P16 gene expression was increased at LD using real-time PCR. (C) Low- and high-density cells were cultured either in osteogenic induction medium or adipogenic induction medium for 7 days. ALP and oil red O staining were conducted at the end of 14 days. ALP staining (upper- and lower-left panels) was induced in the high-density cell group. Oil red O staining (upper- and lower-middle panels) was induced in the low-density cell group. Alizarin red staining (upper- and lower-right panels) was induced in the high-density cell group. Quantification of clones of oil red O staining positive (n=3) is shown in the graph at right. ALP indicates alkaline phosphatase; DAPI, 4′,6-diamidino-2-phenylindole; HD, high density; LD, low density; TIF, telomere dysfunction-induced foci. Paired T-Test, ***, P<.001, data are represented as mean ± SEM. Scale Bar=200μm.
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Research Paper Volume 9, Issue 8 pp 1867-1884
p16(Ink4a) and senescence-associated β-galactosidase can be induced in macrophages as part of a reversible response to physiological stimuli
Relevance score: 5.9485407Brandon M. Hall, Vitaly Balan, Anatoli S. Gleiberman, Evguenia Strom, Peter Krasnov, Lauren P. Virtuoso, Elena Rydkina, Slavoljub Vujcic, Karina Balan, Ilya I. Gitlin, Katerina I. Leonova, Camila R. Consiglio, Sandra O. Gollnick, Olga B. Chernova, Andrei V. Gudkov
Keywords: aging, macrophage, senescent cell, p16(Ink4a), beta-galactosidase
Published in Aging on August 2, 2017
Induction of p16Ink4a and SAβG in macrophages does not require p53. Peritoneal lavage and alginate beads containing SCs (AB) were recovered from wild type (p53+/+) or p53 knockout (p53-/-) mice 15 days after injection (AB model). (A) Representative microphotographs of cryosectioned immunocyte capsules surrounding alginate beads stained with May-Grünwald-Giemsa for histology (10x objective), X-Gal substrate for β-galactosidase activity (SAβG; pH 6.0) (blue) with nuclear fast red counterstain (red), and an immunofluorescent antibody against macrophage marker F4/80 (red) with DAPI nuclear counterstain (blue) (40x objective). (B) Total yield of cells recovered from peritoneal lavage from naïve or AB-injected p53+/+ and p53-/- mouse strains. (C) AB model-elicited immunocyte capsules were pooled equally from 3 mice and p16Ink4a gene expression relative to internal reference gene β2-microglobulin (B2m) was measured by qPCR. (D) β-galactosidase activity from cell extracts of immunocyte capsules from alginate beads recovered from individual mice was measured via 4-MUG hydrolysis, presented as the rate of 4-MU fluorescence (RFU) per minute normalized per microgram of protein. (E) Representative microphotograph of adherence-selected peritoneal lavage from naïve and AB-injected mice stained with X-Gal for SAβG activity. Data show mean ± standard deviation of two independent experiments (n=3 mice per experiment). Statistical comparison between p53+/+ and p53-/- strains are indicated; ns, not significant; **, p-value < 0.01.
Macrophages elicited by alginate-encapsulated SCs possess a modulatable M2-like phenotype. Gene expression analysis of macrophage polarization markers (M1, Nos2 and Il1b; M2, Arg1) of alginate bead model (AB model)-elicited peritoneal macrophages from wild type mice via qPCR. (A) mRNA expression of Nos2 and Arg1 in AB-elicited macrophages adherence-selected from CD11b-enriched peritoneal lavage, as compared to expression in naïve bone marrow-derived macrophages (M0) or following polarization to M1 (IFN-γ for 24 hrs; M1 ctrl) or M2 (IL-4 for 24 hours; M2 ctrl) states. Gapdh expression was used an internal reference gene control. Data shows mean ± standard deviation (n=3). *** p-value < 0.001 compared to M0 control. (B) Peritoneal macrophages elicited by the alginate bead model were treated ex vivo with immunomodulatory agents. qPCR analysis of mRNA expression of indicated genes was normalized to β2-microglobulin (B2m) expression was determined following 72 hour incubation with M1-inducing stimuli (LPS at 1 ng/mL + IFN-γ at 10 ng/mL) or M2-inducing cytokines (IL-4 at 20 ng/mL + IL-13 at 10 ng/mL). Fold change in gene expression following treatment is depicted as mean ± standard deviation relative to non-treated controls; ***, p-value < 0.001. Results are representative of 3 independent experiments with peritoneal lavage cells pooled from at least 3 mice.
Immunomodulatory regulation of p16Ink4a and SAβG in macrophages. Peritoneal lavage cells elicited by alginate-encapsulated SCs from p16Ink4a/Luc mice were treated ex vivo with immunomodulatory agents for 72 hours. (A) p16Ink4a promoter-driven luciferase activity (black bars) and β-galactosidase activity (via 4-MUG hydrolysis) (gray bars) were measured following treatment with M1- and M2-polarizing stimuli: LPS at 1 ng/mL, IFN-γ at 10 ng/mL, LPS/IFN-γ co-treatment, Poly(I:C) at 10 μg/mL, IFN-α at 100 U/mL, IL-4 at 20 ng/mL, IL-13 at 10 ng/ml, and IL-4/IL-13 co-treatment. Results are shown as the mean ± standard deviation for at least 3 independent experiments with statistical significance between treated and non-treated samples depicted. (B) Microphotograph of SAβG-stained adherence-selected macrophages with or without stimulation with LPS (1 ng/mL) for 72 hours (10x objective). (C) mRNA expression of p16Ink4a and β-galactosidase (Glb1) (relative to B2m expression) in macrophages from wild type mice with or without LPS stimulation for 72 hours analyzed via qPCR, as normalized to non-treated controls. Results depicted as mean ± standard deviation (n=3). (D&E) Kinetics of p16Ink4a promoter-driven luciferase activity per cell with or without LPS stimulation (D) or IL-4 stimulation (E), normalized to activity from non-treated cells at time zero. Results are shown as the mean ± standard deviation (n=3). Statistical significance with respect to non-treated control at time zero is indicated. (F) Luciferase activity and β-galactosidase activity (via 4-MUG hydrolysis) from proteose peptone-elicited lavage cells following stimulation with IL-4 (20 ng/mL) for 72 hours, normalized to non-treated controls. Results depicted as mean ± standard deviation (n=3). (G-J) Dose-dependent response of JAK1/2 inhibitor Ruxolitinib (G&H) and STAT6 inhibitor AS1517499 (I&J) on luciferase activity (G&I) and viability via CyQuant Direct assay (H&J) following 72 hours treatment of AB-elicited macrophages in the presence (gray bars) or absence (black bars) of IL-4 (10 ng/mL) stimulation. Results of luciferase activity and viability are representative of two independent experiments, depicted as mean ± standard deviation of data normalized to respective controls lacking inhibitors (with or without IL-4). Luciferase activity and viability are depicted as the percent signal relative to non-treated (NT) controls. Statistical significance between IL-4 stimulated and non-stimulated cells at each concentration of inhibitor is shown. Results are representative of three independent experiments, depicted as mean ± standard deviation. (K) Relative luciferase activity per cell following repolarization of AB-elicited macrophages (via adherence-enriched peritoneal lavage) with M1- and M2-inducing agents. Macrophages were left non-treated (NT) or treated with either LPS (1 ng/ml) or IL-4 (20 ng/ml) for 72 hours (days 1-3), as indicated. For each treatment set, samples were collected at 72 hours (no further treatment; days 4-6 = x). Alternatively, cells were washed and placed in fresh medium (-), medium containing LPS (1 ng/mL) and IFN-γ (10 ng/mL), or medium containing IL-4 (20 ng/mL) and IL-13 (10 ng/mL) and incubated for an additional 72 hours prior to sample collection (as indicated for days 4-6). Luciferase activity is expressed as the percent activity per cell relative to non-treated (NT) controls after the first 72 hours. Results are representative of two independent experiments. *, p-value < 0.05; **, p-value < 0.01; ***, p-value < 0.001.
Elevated p16Ink4a and β-galactosidase is regulated by immunomodulatory agents in macrophages but not mesenchymal SCs. Primary cultures of adipose-derived mesenchymal stromal cells (AdMSC) isolated from p16Ink4a/Luc mice were irradiated (20Gy) and cultured for 10 days for senescence induction. Mock irradiated cells were passaged and used as a proliferating cell control. Response of senescent and proliferating AdMSCs to immunomodulatory agents were compared to that of peritoneal lavage cells elicited by the alginate bead model. (A-C) Characterization of senescent and proliferating AdMSCs. Microphotographs of SAβG-stained cells depicts positive staining of senescent cells, as well as an enlarged and flattened morphology, compared to that of proliferating cell control (A). p16Ink4a promoter-driven luciferase activity (B) and β-galactosidase activity measured via 4-MUG hydrolysis (C) were measured in senescent and proliferating AdMSCs, confirming senescent phenotypes. (D-K) Dose-response curves of LPS (D&E), Poly(I:C) (F&G), IFN-α (H&I), and IFNγ (10 ng/mL), IL-4 (20 ng/mL) and IL-13 (10 ng/mL) (J&K) on p16Ink4a promoter-driven luciferase activity (left panels: D,F,H& J) and β-galactosidase activity measured via 4-MUG hydrolysis (right panels: E,G,I&K) after 72hr treatment. No effect on viability was observed via CyQuant Direct assay (>80% viability). Results are shown as the mean ± standard deviation for at least 3 experiments, with statistical comparison to non-treated controls; *, p-value < 0.05; **, p-value < 0.01; ***, p-value < 0.001. nd, not determined.
Poly(I:C) abrogates elevated p16Ink4a expression in two independent in vivo models. (A-F) p16Ink4a/Luc mice injected with alginate-encapsulated cells (AB injection) were treated with Poly(I:C) in saline at 0, 2 and 10 mg/kg for 3 consecutive days. (A) Schematic representation of alginate bead model experiment depicting timeline and procedures. (B) Representative serial images of mice depicting bioluminescence before and after treatment with 2 mg/kg Poly(I:C). Colored scale depicts relative luminescent signal intensity (in radiance) of minimum and maximum thresholds, as indicated. (C) Graphical representation of bioluminescence (total flux; p/s) measured from the abdomen of treated mice on day 0 (prior to AB injection; blue), day 10 (after AB injection, prior to treatment; red), and day 12 (6 hours after the final treatment; green). Geometric mean is depicted. Statistical significant is calculated with respect to differences between indicated days within treatment groups. (D-F) The effects of Poly(I:C) treatment were analyzed in peritoneal lavage collected within 24 hours of the final treatment of 0, 2, and 10 mg/kg Poly(I:C). Luciferase activity (D), the proportion of peritoneal macrophages (CD45+ CD11b+ CD170- F4/80+ cells) to total lavage cells as quantitated via flow cytometry (E) and β-galactosidase activity (measured via 4-MUG hydrolysis) (F) were quantitated from peritoneal lavage cells. Results are representative of two independent experiments (n=3-6 mice per group per experiment). Statistical significance compared to vehicle-treated controls is depicted; ns, not significant; **, p-value < 0.01; *** p-value < 0.001. (G-M) Chronologically aged mice (83-week old females) were treated with saline or Poly(I:C) (10 mg/kg) for 3 consecutive days. Organs were collected from mice the following day for quantitation of luciferase signal via IVIS. (G) Representative gray-scaled images of organs (1, spleen; 2, lungs; 3, kidneys; 4, perigonadal visceral fat; 5, liver) visualized on IVIS (left) with bioluminescence overlay in color (right). (H) Graphical representation of bioluminescence (total flux; p/s) quantitated from individual organs. Results are representative of two independent experiments (n=3 mice per group per experiment). *, p-value < 0.05. (I-K) Visceral perigonadal adipose tissue was pooled within groups, and the stromal vascular fraction was isolated for analysis. Luciferase activity per cell (I) was measured via luminometer, and the total signal per gram of fat (J) was calculated. (K) The proportion of cells in the SVF expressing macrophage marker F4/80 was measured by detection of immunofluorescent staining via cytometer. (L&M) Microphotographs of whole adipose tissue from mice with or without Poly(I:C) treatment (10 mg/kg) stained for SAβG activity via X-Gal reagent (blue; nuclear fast red counterstain) (L) and immunofluorescent staining of macrophage markers F4/80 (purple), CD11b (red), CD206 (green) and merged overlay with DAPI nuclear counterstain (blue) (M). Results are representative of two independent experiments.
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Research Paper Volume 9, Issue 1 pp 114-132
Induction of fibroblast senescence generates a non-fibrogenic myofibroblast phenotype that differentially impacts on cancer prognosis
Relevance score: 6.5145383Massimiliano Mellone, Christopher J. Hanley, Steve Thirdborough, Toby Mellows, Edwin Garcia, Jeongmin Woo, Joanne Tod, Steve Frampton, Veronika Jenei, Karwan A. Moutasim, Tasnuva D. Kabir, Peter A Brennan, Giulia Venturi, Kirsty Ford, Nicolas Herranz, Kue Peng Lim, James Clarke, Daniel W. Lambert, Stephen S. Prime, Timothy J. Underwood, Pandurangan Vijayanand, Kevin W. Eliceiri, Christopher Woelk, Emma V. King, Jesus Gil, Christian H. Ottensmeier, Gareth J. Thomas
Keywords: tumor microenvironment, myofibroblasts, senescence, collagen, extracellular matrix, senescent fibroblasts
Published in Aging on December 15, 2016
Senescent CAF analyzed ex vivo and in vivo are predominantly SMA-positive. (A) Histogram showing percentage of cells positive for senescence-associated (SA)-β-Galactosidase or SMA-positive stress fiber formation in normal oral fibroblasts (POF) and cancer-associated oral fibroblasts (CAF) grown ex-vivo. Data are presented as Mean ±SEM from 6 POFs and 6 CAF. (B) Representative images of immunohistochemistry on sequential tissue sections of SMA-positive/p16-positive or SMA-positive/p16-negative HNSCC cases. (C) Pie chart showing the percentage of HNSCC cases with stromal staining for SMA or p16. (D) Representative image of double immunofluorescence staining of a p16-positive/SMA-positive HNSCC case showing co-expression of SMA (red) and p16 (green, white arrows; scale bars represent 25µm). (E) Representative immunohistochemistry for SMA and markers of senescence (p53, p21) and oxidative stress (8-OHDG) on sequential tissue sections of HNSCC. See also Supplementary Fig. S2.
Induction of fibroblast senescence generates a myofibroblastic phenotype. HFFF2 fibroblasts were induced to senesce through serial passaging (RS), treatment with γ-irradiation (10Gy; IR) or H2O2 (1mM). Cells were treated with TGF-β1 (2ng/ml) for 72 hours to induce myofibroblast transdifferentiation as a positive control. 4-6 days post stimuli, induction of senescence was confirmed by (A) expression of SA-β-Galactosidase (SA-β-Gal; Scale Bar indicates 100µm) and (B) proliferation assays (cell counts presented as percentage of cells compared to untreated control cells; see also Supplementary Fig. S2A). Cells were examined for myofibroblast features: (C) Representative images of immunofluorescence for SMA expression (green) with DAPI nuclear counterstain (Blue) (Scale Bar indicates 100µm); (D) Western blotting for SMA, palladin and pFAK (HSC-70 as loading control); (E) Representative images of transmission electron microscopy. Arrows highlight sub-membranous microfilaments (Scale Bar indicates 50nm). (F-H) Western blotting for SMA expression (HSC-70 as loading control) and SA-β-Gal quantification following TGF-β1- and irradiation of primary fibroblasts isolated from oral, skin and colonic mucosa respectively. (I) Representative images of collagen gel contraction assays following treatment of HFFF2 with TGF-β1 or different senescence-inducing stimuli. Histogram shows quantification of gel area expressed as the mean ± SEM of 4 replicates; (J) Transwell migration assays with HNSCC (5PT) and EAC (OE33) cell lines. Conditioned media (CM) from HFFF2 fibroblasts induced to senesce through γ-irradiation (10Gy; IR) or to transdifferentiate into myofibroblasts through TGF-β1 was used a chemoattractant in the lower chamber. Data are presented as mean ± SEM and statistics are shown for T-test compared to controls (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). See also Supplementary Fig. S1.
Smad signaling mediates senescence induction of a myofibroblastic phenotype. Western blot showing SMA and phospho(p)-Smad2/3 expression following treatment of HFFF2 fibroblasts with TGF-β1 (A) or irradiation (B) (Smad2 and HSC-70 as loading controls). (C) Representative images of immunofluorescence on HFFF2 fibroblasts showing expression of SMA (green), pSmad2/3 (red), SA-β-Galactosidase activity (grey; in bright field) with DAPI nuclear counterstain (Blue) (scale bar indicates 100µm). (D) Western blots showing SMAD3 knock-down (Tot-FAK as loading control) (left) and SMA expression (HSC-70 as loading control; right) in irradiated HFFF2 cells. (E) Western blot for SMA expression in irradiated HFFF2 pre-treated with a pan TGF-β1 inhibitory antibody. (F) TGFβ-1 assay showing luciferase activity controlled by a TGFβ-1 responsive promoter in MLEC cells co-cultured for 24 hours with untreated or irradiated fibroblasts (y axis indicates the ratio between luciferase activity and HFFF2s protein concentration). Data are presented as mean ± SEM and statistics are shown for T-test compared to controls. See also Supplementary Fig. S3.
Senescence- and TGF-β1-induced myofibroblasts have divergent gene expression profiles. (A-B) RNA-sequencing analysis of HFFF2 cells treated with TGF-β1 (2 ng/ml) or irradiation (10Gy) and grown for 7 days. (A) Unsupervised hierarchical clustering of the expression levels of differentially expressed genes (DEGs; FDR adj. p<0.001), identified using GLM likelihood ratio testing. Expression levels were subjected to Z score scaling within each sample for visualization purposes. Distances were calculated using a Euclidean distance measure. (B) Venn diagram showing the number of DEGs up- or downregulated in TGF-β1 and irradiated fibroblasts compared to controls. (C-E) RT-PCR measurements of mRNA expression levels of genes associated with myofibroblasts (C, E) and senescence (D) in HFFF2 cells used in the RNA-sequencing. Data are presented as mean ±SEM and statistics are shown for t-tests compared to control group. See also Supplementary Fig. S4.
Myofibroblasts and not senescent fibroblasts mediate collagenous ECM deposition. (A) Schematic of experimental procedure for B and C. (B) Representative image of immunofluorescent staining for Fibronectin (FN) in fibroblast-derived matrices (FDM) produced by HFFF2s treated as indicated; FN (orange; pseudo-colored in Fiji) and Dapi (blue) as nuclear counterstain; scale bar represents 50µm. (C) Transwell assay examining OE33 invasion through FDM deposited by HFFF2 fibroblasts induced to transdifferentiate through treatment with TGF-β1 or γ-irradiation (IR). (D-F) Analysis of xenografts formed from 5PT cells injected s.c. into RAG1-/- mice with HFFF2 fibroblasts treated as indicated. (D) Representative images of SMA immunochemistry in 5PT xenografts co-injected with HFFF2s treated as indicated. Histogram shows SMA quantification expressed as % positive area. (E) Representative images of Masson’s trichrome staining for collagen (royal blue) with HFFF2s treated as indicated. Histogram shows quantification expressed as % positive area. (F) Representative images showing multi-photon excitation (MPE) filtered for second harmonic generation to identify collagen fibers on sections from the xenograft tumors as indicated (Scale Bar indicates 100µm). Histogram shows quantification of collagen fiber elongation. Data are presented as mean ± SEM and statistics are shown for T-test compared to controls unless otherwise indicated.
Collagen fiber deposition impacts tumor progression. (A) Gephi network graph where each node represents a gene labelled by color according WGCNA module assignment. Distance between nodes is represented by the TOM connectivity measure. The brown module is the ECM module. (B) ECM module extracted from panel A. (C) Network graph of the ECM module where nodes are color coded according to the correlation with TGF-β1-up DEGs, summarized by a signature eigengene. Red and blue colors show positive and negative correlation, respectively. (D) Correlation of IR-up DEGs with members of the ECM module (color range described above). (E-F) Kaplan-Meier curves showing survival rates in HNSCC patients with greater than average expression of genes associated with myofibroblasts stratified for COL3A1 (E) and CFOG expression (F). (G) Kaplan-Meier curves showing disease specific survival (DSS) rates in HNSCC patients with moderate or high stromal SMA expression (measured by immunohistochemistry), stratified by collagen fiber elongation measured by Second Harmonic Generation imaging. See also Supplementary Fig. S5.
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Research Paper Volume 8, Issue 11 pp 2915-2926
Discovery of piperlongumine as a potential novel lead for the development of senolytic agents
Relevance score: 5.443609Yingying Wang, Jianhui Chang, Xingui Liu, Xuan Zhang, Suping Zhang, Xin Zhang, Daohong Zhou, Guangrong Zheng
Keywords: piperlongumine, aging, senescent cells, senolytic agents, ABT-263, reactive oxygen species, synergistic effect
Published in Aging on November 19, 2016
Senolytic activity of piperlongumine (PL). (A) Structures of PL, 2,3-dihydro-PL, and 7,8-dihydro-PL. (B) Quantification of viable WI-38 non-senescent cells (NC), IR-induced senescent cells (IR-SC), replication-exhausted senescent cells (Rep-SC), or Ras-induced senescent cells (Ras-SC) 72 h after treatment with increasing concentrations of PL (n = 3). (C) Quantification of viable IR-SCs over time after treatment with 10 µM PL (left) or after incubation with 10 µM PL, removal of the drug, and further culture for 72 h (right) (n = 3). (D) Quantification of viable WI-38 NCs and IR-SCs 72 h after treatment with increasing concentrations of 2,3-dihydro-PL or 7,8-dihydro-PL (n = 3). Data are represented as the mean ± SEM.
PL kills SCs by apoptosis. (A) Representative flow cytometric plots to measure apoptotic WI-38 IR-SCs at 48 h after treatment with vehicle (Veh), 10 µM PL, 10 µM Q-VD-Oph (QVD), or the combination of PL and QVD. (B) Quantification of the percentage of viable (gate II: PI− Annexin V−) and apoptotic (gates III and IV: PI− Annexin V+ and PI+ Annexin V+) (right) IR-SCs 48 h after treatment as in (A) (left), and quantification of the percentage of viable IR-SCs 72 h after treatment as in (A) (right). (C) Representative western blot and quantitative analysis of cleaved-poly(ADP-ribose) polymerase (cPARP), procaspase-3 (Procasp-3), cleaved caspase-3 (cCasp-3), and β-actin in NCs and WI-38 IR-SCs 24 h and 48 h after incubation with Veh or 10 µM PL. (D) Representative western blot analysis of RIP1, RIP3, and β-actin in WI-38 NCs and IR-SCs 24 h after incubation with Veh or 10 µM PL. A cell lysate of etoposide-treated Jurkat cells was used as a positive control. Data are represented as the mean ± SEM.
Effect of PL and its analogs on ROS production and senolytic activity in WI-38 IR-SCs. (A) Representative flow cytometric analysis of ROS production in NCs and IR-SCs 24 h after incubation with or without PL by DHR (left) (MFI, mean fluorescence intensity) and quantification of the fold increase of ROS levels in WI-38 NCs and WI-38 IR-SCs cells at the indicated times (middle and right) after incubation with 10 µM PL. As a positive control, cells were treated with 100 µM of H2O2 for 2 h, the H2O2 was removed, and cells were cultured for an additional 24 h (n = 3). (B) Quantification of the fold increase in DHR-123 MFI (left) in WI-38 IR-SCs 24 h after treatment with Veh, 10 µM PL, 2 mM NAC (pretreatment overnight), or the combination of PL and NAC, and (right) the percentage of viable WI-38 IR-SCs 72 h after treatment with Veh, 10 µM PL, 2 mM NAC (pretreatment overnight), or the combination of PL and NAC (n = 3). (C) Structure of PL-NAC and (Left) quantification of viable WI-38 NCs and WI-38 IR-SCs 72 h after treatment with increasing concentrations of PL-NAC (n = 3). (Right) Percentage of 10 µm PL remaining in the culture medium vs. time with or without 2mM NAC. (D) Left panel: quantification of the fold increase in DHR MFI (left) of WI-38 IR-SCs 24 h after treatment with Veh, 10 µM PL, 5 µM γ-tocotrienol (GT3, pretreatment overnight), or the combination of PL and GT3; and right panel: the percentage of viable WI-38 IR-SCs 72 h after treatment with Veh, 10 µM PL, 5 µM GT3 (pretreatment overnight), or the combination of PL and GT3 (n = 3). (E-H) Quantification of the fold increase in DHR-123 MFI after 24 h treatment (left) and viability of WI-38 NCs and WI-38 IR-SCs 72 h treatment (right) after they were treated with increasing concentrations or (E)10 µM BRD4809, (F) 0.5 µM PL-DI, (G) 0.625 µM PL-FPh, and (H) 5 µM PL-7 (n = 3). Data are represented as the mean ± SEM.
PL synergistically and selectively kills SCs in combination with ABT-263. (A) Quantification of NC viability 72 h after the cells incubation with vehicle, 1.25 µM ABT-263, 10 µM PL, or the combination of ABT-263 and PL (n = 3). (B) Quantification of WI-38 IR-SC viability 72 h after incubation with vehicle, 1.25 µM ABT-263, 5 or 10 µM PL, or the combination of ABT-263 and PL (n = 3-5). (C) Quantification of WI-38 IR-SC viability 72 h after incubation with vehicle, 10 µM PL, 0.08-1.25 µM ABT-263, or the combination of ABT-263 and PL (n = 3-6). Data are represented as the mean ± SEM.
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Research Perspective Volume 3, Issue 5 pp 555-563
Embryonic anti-aging niche
Relevance score: 4.6301956Irina M. Conboy, Hanadie Yousef, Michael J. Conboy
Keywords: stem cell aging, regeneration, niche, senescent, cell cycle, Notch, TGF-β, MAPK, muscle, hESC
Published in Aging on May 31, 2011
Young and old myofibers were isolated from hind leg mouse muscle at 3 days post injury by cardiotoxin and were cultured for 24 hours in Ham's F10 supplemented with 10% young or old mouse serum and 50% of the supernatant specified. 10 μM of MEK inhibitor was added to some wells, as indicated. Proliferating muscle progenitor cells that were generated by the activated satellite cells were immunodetected with anti-desmin (green) and anti-BrdU (red) antibodies; Hoechst (blue) was used to label all nuclei. Percent of proliferating myogenic cells was determined by CellProfiler. Typically poor myogenicity of old satellite cells cultured with old serum was rescued by hESC supernatant in a MAPK-dependent manner.
Primary myoblasts were cultured for 24 hours in DMEM + 2% Horse Serum and 50% of the supernatant specified. 10 μM of MEK inhibitor was added to some wells, as indicated. At 24 hours, cells were pulsed with 10 μM BrdU for 2 hours and fixed with 70% ethanol. Cells were immuno-stained for eMyHC (green) and BrdU (red); Hoechst (blue) was used to label all nuclei Automated imaging of these cells was done using ImageXpress and automated counting of percent of eMyHC+ and BrdU+ cells was performed by quantifying at least 100 sites per experimental sample by MetaExpress. hESC supernatant enhanced myoblast proliferation in a MAPK-dependent manner and diminished differentiation into myotubes.
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Research Perspective Volume 3, Issue 2 pp 168-174
Recent developments in the use of γ -H2AX as a quantitative DNA double-strand break biomarker
Relevance score: 4.963023Christophe E. Redon, Asako J. Nakamura, Olga A. Martin, Palak R. Parekh, Urbain S. Weyemi, William M. Bonner
Keywords: DNA damage, DNA repair, γ -H2AX, double-strand break, biomarker, cancer, senescent cells
Published in Aging on February 11, 2011
Because of its sensitivity, the γ-H2AX assay is now utilized in many research areas “from benchtop to bedside” by researchers and clinicians. In addition to being widely used for fundamental research (study of genome stability, DNA repair, etc.) in the last decade, γ-H2AX was identified as a biomarker for cancer (and premalignant lesions) and used to better understand aging. Additionally, γ-H2AX has been developed for radiation biology and biodosimetry for drug development and clinical studies (chemotherapy, the impact of chronic inflammation and diabetes on genome integrity). Finally, γ-H2AX measurement is an efficient and sensitive genotoxic assay for environmental studies.
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Iron retardation in lysosome protects senescent cells from ferroptosis
Relevance score: 7.0832624Yujing Feng, Huaiqing Wei, Meng Lyu, Zhiyuan Yu, Jia Chen, Xinxing Lyu, Fengfeng Zhuang
Keywords: iron accumulation, senescent cells, lysosome, ferroptosis, ferritinophagy
Published in Aging on Invalid Date
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Declined TRB3 expression induces chondrocyte autophagy and senescent in osteoarthritis cartilage
Relevance score: 6.935972Yanqing Gu, Ren Yan, Yang Wang, Yiwen Zeng, Qingqiang Yao
Keywords: TRB3, autophagy, senescent cell, osteoarthritis
Published in Aging on Invalid Date
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Research Paper pp undefined-undefined
Glutaminase inhibitors rejuvenate human skin via clearance of senescent cells: A study using a mouse/human chimeric model
Relevance score: 5.6790442Kento Takaya, Tatsuyuki Ishii, Toru Asou, Kazuo Kishi
Keywords: glutaminase inhibitor, human skin, senescent cell, aging, therapeutic agent
Published in Aging on Invalid Date
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Review pp undefined-undefined
Senescent cell-derived vaccines: a new concept towards an immune response against cancer and aging?
Relevance score: 5.8593297João Pessoa, Sandrina Nóbrega-Pereira, Bruno Bernardes de Jesus
Keywords: cancer, immunotherapy, tumor-associated senescent cells, senescence, antigen, vaccine
Published in Aging on Invalid Date
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Senescent phenotype of astrocytes leads to microglia activation and neuronal death
Relevance score: 5.734361Wenyou Zhang, Xuehan Yang, Jingyue Liu, Yichen Pan, Ming Zhang, Li Chen
Keywords: aging, microglia activation, senescent phenotype of astrocytes, cell-to-cell interaction, neurons
Published in Aging on Invalid Date