Journal: Molecular Medicine Reports

Article Title: Adiponectin inhibits D-gal-induced cardiomyocyte senescence via AdipoR1/APPL1

doi: 10.3892/mmr.2021.12358

Figure Lengend Snippet: Expression of adiponectin, AdipoR1 and APPL1 is downregulated in aged mouse plasma and myocardial tissue. (A) Relative expression levels of p16 and p21 in myocardial tissues of young mice (aged 2 months) and aged mice (aged 15 months) were detected by western blotting. (B) Relative mRNA expression levels of adiponectin, AdipoR1 and APPL1 in the plasma of young mice and aged mice were detected by reverse transcription-quantitative PCR. (C) Relative expression levels of adiponectin, AdipoR1 and APPL1 in myocardial tissues of young mice and aged mice were analyzed using western blot analysis. **P<0.01, ***P<0.001 vs. young mice. AdipoR1, adiponectin receptor 1; APPL1, adaptor protein phosphotyrosine interacting with PH domain and leucine zipper 1.

Article Snippet: The antibodies were as follows: Anti-p16 (1:1,000; cat. no. 18769; Cell Signaling Technology, Inc.), anti-p21 (1:1,000; cat. no. 2947; Cell Signaling Technology, Inc.), anti-adiponectin (1:1,000; cat. no. 2789; Cell Signaling Technology, Inc.), anti-AdipoR1 (1:800; cat. no. bs-0610R; BIOSS), anti-APPL1 (1:1,000; cat. no. 3858; Cell Signaling Technology, Inc.), anti-heme oxygenase (HO)-1 (1:1,000; cat. no. 43966; Cell Signaling Technology, Inc.), anti-high mobility group box 1 (HMGB1; 1:1,000; cat. no. 3935; Cell Signaling Technology, Inc.) and mouse anti-rabbit secondary antibody (1:1,000; cat. no. 5127; Cell Signaling Technology, Inc.).

Techniques: Expressing, Clinical Proteomics, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Molecular Medicine Reports

Article Title: Adiponectin inhibits D-gal-induced cardiomyocyte senescence via AdipoR1/APPL1

doi: 10.3892/mmr.2021.12358

Figure Lengend Snippet: Expression of adiponectin, AdipoR1 and APPL1 is downregulated in D-gal-treated cardiomyocytes. (A) Relative expression levels of p16 and p21 in H9c2 cells treated with 2.5, 5 and 10 g/l of D-gal were analyzed using western blot analysis. *P<0.05, **P<0.01, ***P<0.001 vs. control. Transfection with Ov-adiponectin promotes the expression of AdipoR1 and APPL1 in D-gal-treated cardiomyocytes. (B) Relative mRNA expression of adiponectin in H9c2 cells before and after transfection with Ov-adiponectin plasmid was analyzed via RT-qPCR. ***P<0.001 vs. control; ### P<0.001 vs. D-gal + Ov-NC. (C) Relative protein expression of adiponectin in H9c2 cells before and after transfection with Ov-adiponectin plasmid was analyzed using RT-qPCR. ***P<0.001 vs. control; ## P<0.01 vs. D-gal + Ov-NC. (D) Relative mRNA expression levels of AdipoR1 and APPL1 in H9c2 cells before and after transfection with Ov-adiponectin plasmids were detected using RT-qPCR. ***P<0.001 vs. control; ### P<0.001 vs. D-gal + Ov-NC. AdipoR1, adiponectin receptor 1; APPL1, adaptor protein phosphotyrosine interacting with PH domain and leucine zipper 1; RT-qPCR, reverse transcription-quantitative PCR; NC, negative control; D-gal, D-galactose; Ov-, overexpression.

Article Snippet: The antibodies were as follows: Anti-p16 (1:1,000; cat. no. 18769; Cell Signaling Technology, Inc.), anti-p21 (1:1,000; cat. no. 2947; Cell Signaling Technology, Inc.), anti-adiponectin (1:1,000; cat. no. 2789; Cell Signaling Technology, Inc.), anti-AdipoR1 (1:800; cat. no. bs-0610R; BIOSS), anti-APPL1 (1:1,000; cat. no. 3858; Cell Signaling Technology, Inc.), anti-heme oxygenase (HO)-1 (1:1,000; cat. no. 43966; Cell Signaling Technology, Inc.), anti-high mobility group box 1 (HMGB1; 1:1,000; cat. no. 3935; Cell Signaling Technology, Inc.) and mouse anti-rabbit secondary antibody (1:1,000; cat. no. 5127; Cell Signaling Technology, Inc.).

Techniques: Expressing, Western Blot, Control, Transfection, Plasmid Preparation, Quantitative RT-PCR, Reverse Transcription, Real-time Polymerase Chain Reaction, Negative Control, Over Expression

Journal: Molecular Medicine Reports

Article Title: Adiponectin inhibits D-gal-induced cardiomyocyte senescence via AdipoR1/APPL1

doi: 10.3892/mmr.2021.12358

Figure Lengend Snippet: Adiponectin inhibits D-gal-induced cardiomyocyte senescence via AdipoR1/APPL1. (A and B) Relative expression levels of AdipoR1 in D-gal-treated H9c2 cells with Ov-adiponectin before and after transfection with si-AdipoR1 and si-APPL were measured using RT-qPCR. **P<0.01, ***P<0.001 vs. D-gal + Ov-adiponectin. (C and D) Relative expression levels of APPL1 in D-gal-treated H9c2 cells with Ov-adiponectin before and after transfection with si-AdipoR1 and si-APPL were measured using RT-qPCR. ***P<0.001 vs. D-gal + Ov-adiponectin. (E) Cellular senescence in the different groups was detected using SA-β-gal staining. (F) Relative expression levels of p16 and p21 in the different groups were detected using western blot analysis. ***P<0.001 vs. control; ## P<0.01, ### P<0.001 vs. D-gal + Ov-NC; $ P<0.05 vs. D-gal + Ov-adiponectin + negative-NC. AdipoR1, adiponectin receptor 1; APPL1, adaptor protein phosphotyrosine interacting with PH domain and leucine zipper 1; Ov-, overexpression; RT-qPCR, reverse transcription-quantitative PCR; NC, negative control; si-, small interfering RNA; D-gal, D-galactose; SA-β-gal, senescence-associated β-galactose.

Article Snippet: The antibodies were as follows: Anti-p16 (1:1,000; cat. no. 18769; Cell Signaling Technology, Inc.), anti-p21 (1:1,000; cat. no. 2947; Cell Signaling Technology, Inc.), anti-adiponectin (1:1,000; cat. no. 2789; Cell Signaling Technology, Inc.), anti-AdipoR1 (1:800; cat. no. bs-0610R; BIOSS), anti-APPL1 (1:1,000; cat. no. 3858; Cell Signaling Technology, Inc.), anti-heme oxygenase (HO)-1 (1:1,000; cat. no. 43966; Cell Signaling Technology, Inc.), anti-high mobility group box 1 (HMGB1; 1:1,000; cat. no. 3935; Cell Signaling Technology, Inc.) and mouse anti-rabbit secondary antibody (1:1,000; cat. no. 5127; Cell Signaling Technology, Inc.).

Techniques: Expressing, Transfection, Quantitative RT-PCR, Staining, Western Blot, Control, Over Expression, Reverse Transcription, Real-time Polymerase Chain Reaction, Negative Control, Small Interfering RNA

Journal: bioRxiv

Article Title: Elimination of senescent cells with senolytic host-directed therapy reduces tuberculosis progression in mice

doi: 10.1101/2025.03.28.645957

Figure Lengend Snippet: (a) Schematics of the experimental strategy to measure senescence markers (p21, p16 and SA-βGal activity) and SASP cytokines levels in mice lungs at 10 and 20 days p.i. Day 1 CFU= 55. (b) %p21+p16+ and (c) %SA-βGal+ (CellEvent Senescence green+) cells out of all live lung (uninfected and Mtb- infected mice) and Spleen ( Mtb- infected mice) cells at 10 days p.i. as determined by multicolor flow cytometry. (d) %p21+p16+ and (e) %SA-βGal+ cells out of different lung-cell types at 10 days p.i., as determined by multicolor flow cytometry. Gating for p16⁺, p21⁺, and SA-βGal⁺ events for each cell type were established using reference cells from Mtb -infected WT B6 mice (set at 1 %). (AMs: Alveolar macrophages, IMs: Interstitial macrophages) (f) Normalized concentration of SASP-cytokines in lung homogenates at 20 dpi and 4 wpi as measured by a LEGENDplex™ Mouse Inflammation Panel (13-plex). The data are means ± SEM. Each data point represents a mouse (n=11-12). (two-way ANOVA with Tukey’s (b-e) or Dunnett’s (f) multiple comparisons test).

Article Snippet: The PVDF membranes were blocked using 5% Blotting-grade blocker (Bio-Rad; 1706404) in TBST buffer-Tris-buffered saline (Quality Biological; 351086101)+ 0.1% Tween 20 (Sigma; P2287) for 1-2 hour and incubated overnight at 4°C with the primary antibodies from Mouse Reactive Senescence Marker Antibody Sampler Kit (Cell Signaling Technology-78551)-Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb (1: 1000 dilution), Lamin B1 (E6M5T) Rabbit mAb (1: 1000 dilution), HMGB1 (D3E5) Rabbit mAb (1: 1000 dilution), p16 INK4A (E5F3Y) Rabbit mAb (1: 1000 dilution) and β-Actin (D6A8) Rabbit mAb (Cell Signaling Technology; 8457) (1:5000 dilution).

Techniques: Activity Assay, Infection, Flow Cytometry, Concentration Assay

Journal: bioRxiv

Article Title: Elimination of senescent cells with senolytic host-directed therapy reduces tuberculosis progression in mice

doi: 10.1101/2025.03.28.645957

Figure Lengend Snippet: (a) Representative H&E-stained images of Mtb -infected mice lungs treated with Vehicle or drugs, and respective ImageJ-based quantification. Yellow arrow indicates necrotic granuloma in Vehicle treated B6.Sst1S mice at 5 wpi. The data are means ± SEM. Each data point represents a mouse. Statistical analysis between two groups was done by unpaired two-tailed Student’s t test. Square represents necrotic granuloma formation in the lung. (b) Representative γH2A.X-Immunohistochemistry images of mice after lungs vehicle/ drugs treatment, and respective Violin plots to show ImageJ quantification of γH2A.X-stained area. Each data point represents a mouse. Statistical analysis between two groups was done by unpaired two-tailed Student’s t test. (c) %p21+βGal+ and %p16+βGal+ subpopulation observed in all live lung cells of Mtb -infected mice (n= 5-8). The data are means ± SEM. Each data point represents a mouse. Statistical analysis was calculated by two-way ANOVA with Dunnett’s multiple comparisons test. Bubble plot to show %p21+βGal+ and %p16+βGal+ subpopulation in different lung cell types in Mtb -infected (d) B6.Sst1S mice (n= 8) and (e) WT B6 old mice (n= 5) at indicated time point and treatment groups. The size of the bubble indicates %subpopulation out of all of particular cell types and color is adjusted p values relative to Veh group. Statistics are calculated by one-way ANOVA with Tukey’s multiple comparisons test ( p > 0.15: ns).

Article Snippet: The PVDF membranes were blocked using 5% Blotting-grade blocker (Bio-Rad; 1706404) in TBST buffer-Tris-buffered saline (Quality Biological; 351086101)+ 0.1% Tween 20 (Sigma; P2287) for 1-2 hour and incubated overnight at 4°C with the primary antibodies from Mouse Reactive Senescence Marker Antibody Sampler Kit (Cell Signaling Technology-78551)-Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb (1: 1000 dilution), Lamin B1 (E6M5T) Rabbit mAb (1: 1000 dilution), HMGB1 (D3E5) Rabbit mAb (1: 1000 dilution), p16 INK4A (E5F3Y) Rabbit mAb (1: 1000 dilution) and β-Actin (D6A8) Rabbit mAb (Cell Signaling Technology; 8457) (1:5000 dilution).

Techniques: Staining, Infection, Two Tailed Test, Immunohistochemistry

Journal: Biogerontology

Article Title: Anti-SASP and anti-inflammatory activity of resveratrol, curcumin and β-caryophyllene association on human endothelial and monocytic cells

doi: 10.1007/s10522-021-09915-0

Figure Lengend Snippet: Characterization of endothelial cells in RS- and IS-HUVEC models. a Growth curve of a pool of HUVECs—Y-axis: Cumulative Population Doubling (cPD); X-axis: cell passages from P1 to P22 (i); two representative pictures of SA-β-Gal staining of young (ii) and senescent (iii) HUVECs; % of SA- β-Gal (iv). Cells with SA-β-Gal < 10% were considered young cells (yHUVEC), while those with SA- β-Gal > 60% were identified as senescent cells; b representative picture of SA- β-Gal-positive cells (i), dose–response curve (ii) and % of SA- β-Gal (iii) after treatment with 5 nM (left image, 50 nM (central image) and 100 nM (right image) of Doxorubicin; c relative expression of p16 ink4a mRNA in yHUVEC (P4), RS-HUVECs (P21) and IS-HUVECs (Doxorubicin 50 nM); d densitometric analysis of p16 ink4a protein level in RS-HUVEC and IS-HUVEC. Histograms represent the mean of the protein level and the relative expression measured in three different experiments ± SD. Paired t test, *p < 0.05 versus yHUVEC. RS replicative senescence, IS induced senescence

Article Snippet: Mouse anti-SIRT1 (Abcam), rabbit anti-Caspase-1 p10 (Santa Cruz Biotechnology), mouse anti-p16 ink4a (Santa Cruz) and, rabbit anti-α-tubulin (Cell Signaling), were used as primary antibodies.

Techniques: Staining, Expressing

Journal: Biogerontology

Article Title: Anti-SASP and anti-inflammatory activity of resveratrol, curcumin and β-caryophyllene association on human endothelial and monocytic cells

doi: 10.1007/s10522-021-09915-0

Figure Lengend Snippet: p16 ink4a and SIRT1 protein level after treatment with bCUR, RSV and BCP and their combination in RS- and IS-HUVECs. a and c p16 ink4a protein level and densitometric analysis; b and d SIRT1 protein level and densitometric analysis. Data are reported as fold change vs untreated senescent HUVECs. All data were normalized using α-tubulin as internal control. Bands were quantified by ImageJ; histograms represent the mean of the protein expression detected in three different experiments ± SD. Paired t test *p < 0.05 versus RS- and IS-HUVECs; **p < 0.01 versus RS- and IS-HUVECs. bCUR, bioCurcumin; RSV, resveratrol; BCP, β‐caryophyllene; RS, replicative senescence; IS, induced senescence. RS-HUVECs data are depicted in the left panel (solid-coloured histograms); IS-HUVECs data are reported in the right panel (striped histograms)

Article Snippet: Mouse anti-SIRT1 (Abcam), rabbit anti-Caspase-1 p10 (Santa Cruz Biotechnology), mouse anti-p16 ink4a (Santa Cruz) and, rabbit anti-α-tubulin (Cell Signaling), were used as primary antibodies.

Techniques: Control, Expressing

Journal: Biogerontology

Article Title: Anti-SASP and anti-inflammatory activity of resveratrol, curcumin and β-caryophyllene association on human endothelial and monocytic cells

doi: 10.1007/s10522-021-09915-0

Figure Lengend Snippet: Summarized data of the anti-SASP and anti-inflammatory effect of bCUR, RSV and BCP alone and in combination (MIX) on RS- HUVECs, IS-HUVECs and THP-1 cells

Article Snippet: Mouse anti-SIRT1 (Abcam), rabbit anti-Caspase-1 p10 (Santa Cruz Biotechnology), mouse anti-p16 ink4a (Santa Cruz) and, rabbit anti-α-tubulin (Cell Signaling), were used as primary antibodies.

Techniques:

Journal: bioRxiv

Article Title: Progressive Senescence Programs Induce Intrinsic Vulnerability to Aging-related Breast Cancer

doi: 10.1101/2023.05.08.539818

Figure Lengend Snippet: (A) Diagram showing the workflow of the transcription factors enrichment analysis for each cell state using the ENCODE, ChEA, and TRRUST database. Reliable candidates of transcription factors regulating each state were supported by at least two databases. (B) Table showing enriched transcription factors based on differentially expressed genes in each cell state. Black lines indicate TFs enrichment in two databases. (C) Activity score of transcription factors enriched in e-Sen state presented by target gene index over the pseudotime. We used the differentially expressed genes in each cell state, and inferred the transcription factors with the CHIP-seq target database. If the targets of certain TF factor are enriched in specific cell state, the TF factor will be popped out as a signature factor. (D) Representative β-gal staining images of mammary gland from young (3 month), aged (17 month) and K14 - Cre Bcl11b fl/fl (3 month) mice showing senescent cells. Scale bar, 20μm. (E) Percentage of p16 Ink4a (scale bar, 20μm) positive cells in mammary epithelial cells from young (4 month), old (22 month), control K14 - Cre Bcl11b wt/wt (4 month) and K14 - Cre Bcl11b fl/fl (4 month) mice. Statistical analysis was performed using two-tailed unpaired t-tests; data are presented as mean ± SD; ***P<0.001, ****P<0.0001. (F) Relative basal luminal proportion of mammary gland epithelia in young (n=54) and old (n=13) mice. Statistical significance was determined by two-tailed unpaired t-tests; data are presented as mean ± SD; ****P<0.0001. (G) Quantification of relative basal/luminal proportion in mammary epithelia in K14 - Cre Bcl11b fl/fl mTmG reporter mice (n=9). GFP+ cells were regarded as Bcl11b KO cells while tdTomato+ cells were regarded as WT cell control in the same gland. Statistical significance was determined by two-tailed unpaired t-tests; data are presented as mean ± SD; ****P<0.0001. (H-I) Density map (H) and percentage (I) of mammary cell states in each age group and K14 - Cre Bcl11b fl/fl group. (J) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showing pathways significantly enriched in K14 - Cre Bcl11b fl/fl CD49f high EpCAM low cells. (K) Boxplots showing the scEntropy score of K14 - Cre Bcl11b fl/fl CD49f high EpCAM low cells increased to a level similar to l-Sen cells. The interquartile (box limits) and median (center blank line). (L) Comparison of SASP gene score in WT and K14 - Cre Bcl11b fl/fl CD49f high EpCAM low cells. The box plots show the interquartile (box limits) and median (center blank line). Statistical analysis was performed using wilcoxon test.

Article Snippet: Sections were blocked with TBS+0.1% Triton X-100+2% BSA+10% Donkey serum for 1 hr at RT, and then stained with primary antibody mouse anti-p16 Ink4a (Santa Cruz, sc-1661), Rb anti-p65 (Cell signaling technology, 8242), Rat anti-Bcl11b (Abcam, ab18465) 1:200 overnight at 4°C.

Techniques: Activity Assay, ChIP-sequencing, Staining, Control, Two Tailed Test, Comparison

Journal: bioRxiv

Article Title: Progressive Senescence Programs Induce Intrinsic Vulnerability to Aging-related Breast Cancer

doi: 10.1101/2023.05.08.539818

Figure Lengend Snippet: (A) Representative immunofluorescence staining of p16 Ink4a (scale bar, 20μm) in mammary epithelial cells from young (4 month), old (22 month), control K14 - Cre Bcl11b wt/wt (4 month) and K14 - Cre Bcl11b fl/fl (4 month) mice. (B) Representative FACS plots of basal cells and luminal cells in young (2m) and old (20m) mammary gland lineage-population. (C) FASC analysis of mammary epithelia in K14 - Cre Bcl11b fl/fl mTmG mice showing the relative proportion of basal cells and luminal cells. (D) Representative wholemount staining and duct width quantifications of young (2m-5m) and aged (12m-29m) mammary glands. Scale bar, 500μm; statistical analysis was performed using two-tailed unpaired t-tests; data are presented as mean ± SD; ****P<0.0001. (E) Schematic diagram for the workflow of the ageing phenotype quantification for WT and K14 - Cre Bcl11b fl/fl mammary cells after multiple rounds of reproduction cycles. Basically, WT and K14 - Cre Bcl11b fl/fl mammary cells were transplanted onto cleared fat pads. Recipient mice were mated with male mice for 3 rounds. Whole mount and colony formation assays were performed after the last round of pregnancy. (F-H) Representative pictures of mammary wholemounts (F; Scale bar, 500μm), HE staining (G; Scale bar, 200μm) and colony formation (H; Scale bar, 1000μm) of WT and K14 - Cre Bcl11b fl/fl transplanted mammary glands after multiple rounds of pregnancy. Statistical analysis was performed using two-tailed unpaired t-tests; data are presented as mean ± SD; *P<0.05. (I) ELDA plot of limiting dilution transplant of young (3m) and old (18m) mammary gland lineage-cells. P<0.0001. (J) Table for limiting dilution transplant of young (3month) and old (18 month) mammary gland lineage-cells.

Article Snippet: Sections were blocked with TBS+0.1% Triton X-100+2% BSA+10% Donkey serum for 1 hr at RT, and then stained with primary antibody mouse anti-p16 Ink4a (Santa Cruz, sc-1661), Rb anti-p65 (Cell signaling technology, 8242), Rat anti-Bcl11b (Abcam, ab18465) 1:200 overnight at 4°C.

Techniques: Immunofluorescence, Staining, Control, Two Tailed Test

Journal: bioRxiv

Article Title: Procyanidin C1 is a natural agent with senolytic activity against aging and age-related diseases

doi: 10.1101/2021.04.14.439765

Figure Lengend Snippet: ( a ) Measurement of senescent cell survival by SA-β-Gal staining assay. Procyanidin C1 (PCC1) was applied at increasing concentrations. CTRL, proliferating. SEN, senescent. ( b ) Senolytic activity appraisal by calculating the percentage of surviving senescent cells induced by BLEO at increasing PCC1 concentrations. ( c ) Apoptotic assay of cells treated by PCC1 by examination of caspase 3/7 activity. ( d ) Representative images of SA-β-Gal staining upon treatment with vehicle or PCC1. CTRL, proliferating cells. TIS, therapy-induced senescence (by BLEO). RS, replicative senescence. OIS, oncogene-induced senescence (by oncogenic HRas G12V ). Scale bar, 20 μm. ( e ) FACS measurement of CTRL and SEN cells after processing with an annexin V-FITC/PI kit and DAPI staining to determine apoptosis. ( f ) Comparative quantification of the percentage of viable (Q4: PI − /annexin V − ) and apoptotic (Q2 and Q3: PI + /annexin V + and PI − /annexin V + , respectively) cells in the CTRL or SEN populations treated with vehicle or GSE for 3 days (n = 3). ( g ) Immunofluorescence staining of PSC27 cells. RS was induced by consecutive proliferation, before cells were treated by PCC1. Red, p16 INK4a . Cells at an early passage (p10) were used as a negative control. The senolytic agent ABT-263 was employed as a positive control. Scale bar, 20 μm. ( h ) Statistics of immunofluorescence staining positivity in assays described in ( g ). ( i ) Examination of PCC1-induced senolytic activity by pan-caspase inhibition (20 μM QVD-OPh). ABT-263 used as a positive senolytic control. ( j ) Population doubling assessment assay upon different treatments. PCC1 was applied on the 8 th day after the beginning of experiments as indicated. In experiments of c - j , PCC1 was used at 100 μm. Data are shown as mean ± SD and representative of 3 biological replicates. ^, P > 0.05. *, P < 0.05. **, P < 0.01. ***, P < 0.001. ****, P < 0.0001.

Article Snippet: Slides were retrieved for 20□min using Epitope Retrieval 1 (Citrate; Leica) and incubated in Protein Block (Dako) for 5 min. Primary antibodies were diluted in Background Reducing Diluent (Dako) as follows: p16 INK4A (rabbit, monoclonal; abcam, Cambridge, MA, catalog no. ab108349) at 1:600, cleaved-caspase 3 (rabbit, polyclonal; Cell Signaling, catalog no. 9661L) at 1:200, and F4/80 (rat, monoclonal; abcam, catalog no. ab90247) at 1:500, except for CD68 antibody (mouse, monoclonal; Dako, catalog no. M0876), which was diluted in Bond Diluent (Leica) at 1:200.

Techniques: Staining, Activity Assay, Immunofluorescence, Negative Control, Positive Control, Inhibition, Control

Journal: eLife

Article Title: Senotherapeutic drugs for human intervertebral disc degeneration and low back pain

doi: 10.7554/eLife.54693

Figure Lengend Snippet: ( A ) Representative images indicating how we deemed positive and negative p16 INK4a ( a ), Ki-67 ( d ), and caspase-3 ( g ) staining. ( b, e and h ) Magnified images of ( a , d and g ). Arrow heads indicate positive (green) and negative (red) staining. ( c, f and i ) no secondary antibody control photomicrographs. Quantification of ( B ) p16 INK4a (n = 7), ( C ) Ki-67, and ( D ) caspase-3 expression, (n = 8). Scale bars: 20 μm in ( A ). The cells were from degenerating IVDs as indicated in . Values are presented as mean ± SEM in ( B–D ). * Indicates a significant difference assessed by the two-tailed Student's t-test: p<0.05; **p<0.01 and ****p<0.0001. Figure 1—source data 1. Excel file containing the percentage of p16 Ink4a , ki-67 and caspase three positive in NP and AF cells control and treated with RG-7112.

Article Snippet: Antibody , Mouse monoclonal Anti- p16 Ink4a , Abcam , ab54210 RRID: AB_2059963 , IHC-P (1:500).

Techniques: Staining, Control, Expressing, Two Tailed Test

Journal: eLife

Article Title: Senotherapeutic drugs for human intervertebral disc degeneration and low back pain

doi: 10.7554/eLife.54693

Figure Lengend Snippet: ( A ) Metabolic activity in control and treated NP and AF pellet cultures from degenerate IVDs (n = 6). ( B ) Representative photomicrographs of untreated ( a–c ) and RG-7112 treated ( d–f ) NP cells stained for cytocalcein (viable), apopxin (apoptotic) and merged images. ( C ) Quantification of apoptosis in treated and control groups (n = 3). ( D ) p16 INK4a separate-staining with Ki-67 ( a–c ) and colocalization with caspase-3 ( d–f ) in NP cells (n = 3). ( E ) RG-7112 (5 μM) selectively induced apoptosis in degenerate NP cells compared to non-mildly-degenerate as measured by the caspase 3/7 activity kit. The results are expressed as a percentage compared to the untreated control (n = 8). ( F ) Alamar blue assay comparison of the effect of RG-7112 (5 μM) on the viability of degenerate and non-degenerate NP cells. Results are presented as fold-change compared to the control (n = 8). ( G ) Glycosaminoglycan (sGAG) levels in the NP and AF-treated pellet media at days 7, 14, 21 and 28 were assessed by DMMB. GAG release was normalized to day 0 GAG concentration and then to the sGAG concentration of untreated culture media (n = 6). Scale bars: 50 μm in ( B ) and 25 μm ( D ). Values are presented as mean ± SEM in ( A, C, E, F and F ). Data was analyzed by Student’s t-tests to compare two groups. * indicates a significant difference of p<0.05, ** of p<0.01 and *** of p<0.001. Figure 1—figure supplement 1—source data 1. Excel file containing the fluorescence values for metabolic and caspase3/7 activity, the number of apoptotic cells, and the concentrations of sGAG in NP (and AF) cells treated and untreated with RG-7112.

Article Snippet: Antibody , Mouse monoclonal Anti- p16 Ink4a , Abcam , ab54210 RRID: AB_2059963 , IHC-P (1:500).

Techniques: Activity Assay, Control, Staining, Alamar Blue Assay, Comparison, Concentration Assay, Fluorescence

Journal: eLife

Article Title: Senotherapeutic drugs for human intervertebral disc degeneration and low back pain

doi: 10.7554/eLife.54693

Figure Lengend Snippet: ( A ) Schematic of the ex vivo organ culture experiment. Lumbar spines from organ donors were assessed radiographically for signs of degeneration. Three discs per experiment were isolated from the same spine, cultured for 4–6 days then scanned with MRI and injected with vehicle, o-Vanillin or RG-7112. Discs were then cultured for an additional 28 days, with media changes every 3–4 days. The discs were scanned by MRI again at day 28. Media and tissues were used for SASP factor release and histology respectively. ( B ) Representative images of mid-axial T1ρ slices pre-treatment ( a–b, e–f, i–j ) and the same location post treatment ( c–d, g–h, k–l ) with vehicle (CTRL), RG-7112 (5 μM) or o-Vanillin (100 μM). The heatmap correlates the red color with the highest and the blue color with the lowest T1ρ values. ( C ) Quantification for NP regions with the graph showing percentage change in T1ρ values post- compared to the pre-treatment scans. ( D ) Representative safranin O/fast green staining of histological sections. ( E ) Representative images of disc sections stained with antibodies against p16 INK4a and Ki-67. Quantification of ( F ) p16 INK4a and ( G ) Ki-67 expression. Scale bars = 150 µm in 4D, 25 µm in 4E (p16 INK4a ) and 50 µm in 4E (ki-67); Error bars represent mean ± SEM, Statistical significance was assessed by two-tailed Student's t-test to compare pre and post disc groups ( C ) and by repeated measures Analysis of Variance (ANOVA) with Turkey’s post hoc test for multiple pairwise comparison in ( F and G ). *Indicates p<0.05 and **indicates p<0.01, n = 4 for each condition. The tissues were from degenerating IVDs as indicated in and . Figure 4—source data 1. Excel file containing the complete list of donors, slices and their respective voxel intensities, p16 Ink4a and ki-67-positive cells in control and treated discs.

Article Snippet: Antibody , Mouse monoclonal Anti- p16 Ink4a , Abcam , ab54210 RRID: AB_2059963 , IHC-P (1:500).

Techniques: Ex Vivo, Organ Culture, Isolation, Cell Culture, Injection, Staining, Expressing, Two Tailed Test, Comparison, Control

Journal: eLife

Article Title: Senotherapeutic drugs for human intervertebral disc degeneration and low back pain

doi: 10.7554/eLife.54693

Figure Lengend Snippet:

Article Snippet: Antibody , Mouse monoclonal Anti- p16 Ink4a , Abcam , ab54210 RRID: AB_2059963 , IHC-P (1:500).

Techniques: Ab Array, Activity Assay, cDNA Synthesis, Gene Expression, Luminex, Software, Staining

Journal: EBioMedicine

Article Title: p53 Modulates the Fate of Cardiac Progenitor Cells Ex Vivo and in the Diabetic Heart In Vivo

doi: 10.1016/j.ebiom.2017.01.028

Figure Lengend Snippet: p53, cardiomyocytes and CPCs. (a, b) Ki67-positive (a) and apoptotic TUNEL-positive (b) cardiomyocytes in young-adult, 8–11 months (WT: n = 9; p53-tg: n = 7), and old, 20–25 months (WT: n = 6; p53-tg: n = 8), WT and p53-tg mice. * p < 0.05 vs. young-adult WT; ** p < 0.05 vs. old WT; *** p < 0.05 vs. young-adult p53-tg. (c) p16 INK4a -positive cardiomyocytes in old, 18–33 months, WT ( n = 4) and p53-tg ( n = 9) mice. (d, e) Number of c -kit-positive CPCs in atrial myocardium (d) and fraction of cycling Ki67-positive CPCs (e). WT: n = 3; p53-tg: n = 4. (f) Population doubling time (PDT) in WT-CPCs (WT; n = 3) and p53-tg-CPCs (p53-tg; n = 3). (g) Fraction of Ki67 labeled WT-CPCs ( n = 3) and p53-tg-CPCs ( n = 3). (h) Fraction of p16 INK4a labeled WT-CPCs ( n = 3) and p53-tg-CPCs ( n = 3). (i) Apoptosis of WT-CPCs ( n = 3) and p53-tg-CPCs ( n = 3) measured by Annexin V assay. In all cases data are shown as mean ± SD. * p < 0.05 vs. WT.

Article Snippet: The following antibodies were utilized: mouse monoclonal anti -p53 (Cell Signaling), rabbit polyclonal anti -p53 (Ser 37) (Cell Signaling Technology: cat. no. 2524, RRID: AB_331743 ), rabbit polyclonal anti -p53 (Ser15) (Cell Signaling Technology: cat. no. 9286S, RRID: AB_331741 ) and mouse monoclonal anti -p16 INK4a (Cell Signaling Technology: cat. no. 8884S, RRID: AB_11129865 ).

Techniques: TUNEL Assay, Labeling, Annexin V Assay

Journal: EBioMedicine

Article Title: p53 Modulates the Fate of Cardiac Progenitor Cells Ex Vivo and in the Diabetic Heart In Vivo

doi: 10.1016/j.ebiom.2017.01.028

Figure Lengend Snippet: p53 favors the functional recovery of CPCs from oxidative stress in vitro. (a) Western blotting of p16 INK4a at baseline, after Doxo-pulse and following recovery of WT-CPCs (WT) and p53-tg-CPCs (p53-tg); n = 3 in all cases. Optical density data are mean ± SD. * p < 0.05 vs. WT-Control. ** p < 0.05 vs. WT-Doxo-pulse. *** p < 0.05 vs. WT-recovery. (b) p16 INK4a labeling (upper left panel, yellow) of WT-CPCs exposed to Doxo. Nuclei are stained by DAPI (upper right panel, blue). Phalloidin (lower left panel, white). Merge of p16 INK4a , DAPI and phalloidin (lower right panel). Scale bar, 50 μm. Fraction of p16 INK4a -positive WT-CPCs and p53-tg-CPCs at baseline, following Doxo-pulse and after recovery; n = 3 in all cases. Data are mean ± SD. * p < 0.05 vs. WT-Control. ** p < 0.05 vs. WT-Doxo-pulse. *** p < 0.05 vs. WT recovery. † p < 0.05 vs. p53-tg control. ‡ p < 0.05 vs. p53-tg Doxo-pulse. (c) Number of DDR foci in WT-CPCs and p53-tg-CPCs at baseline, after Doxo-pulse and following recovery; n = 3 in all cases. For statistics see panel B. (d) Nucleoids in WT-CPCs and p53-tg-CPCs at baseline, following Doxo-pulse and after recovery are stained with Vista green dye (green). Comets are apparent in Doxo-pulse and after recovery of WT-CPCs, while intact DNA is noted in p53-tg-CPCs after recovery. (e) Damaged DNA in nuclei of WT-CPCs and p53-tg-CPCs at baseline, after Doxo-pulse and following recovery; n = 3 in all cases. For statistics see panel B. (f) Fraction of Ki67-positive WT-CPCs and p53-tg-CPCs following 24, 48 and 72 h recovery period; n = 3 in all cases.* p < 0.05 vs. 24 h. ** p < 0.05 vs. 48 h.

Article Snippet: The following antibodies were utilized: mouse monoclonal anti -p53 (Cell Signaling), rabbit polyclonal anti -p53 (Ser 37) (Cell Signaling Technology: cat. no. 2524, RRID: AB_331743 ), rabbit polyclonal anti -p53 (Ser15) (Cell Signaling Technology: cat. no. 9286S, RRID: AB_331741 ) and mouse monoclonal anti -p16 INK4a (Cell Signaling Technology: cat. no. 8884S, RRID: AB_11129865 ).

Techniques: Functional Assay, In Vitro, Western Blot, Control, Labeling, Staining

Journal: Cardiovascular Research

Article Title: Efficacy and limitations of senolysis in atherosclerosis

doi: 10.1093/cvr/cvab208

Figure Lengend Snippet: Senescence markers in primary human and mouse VSMCs undergoing senescence. ( A ) % EdU + in cultured human VSMCs (Control), after 24h treatment with 500 nM doxorubicin (Dox 1d), after an additional 21 days recovery in control conditions (control 21d) or after doxorubicin (Dox 1d+ 21d), or at replicative senescence (RS). ( B – D ) mRNA levels of Lamin B1, p16, and p21 in cell populations described in ( A ) relative to control (1d) cells. ( E ) Western blot for Lamin B1, p16, p21, and p53 for cells treated in ( A ). n = 6–8 human VSMC isolates. ( F and G ) EdU + % ( F ) or SAβG + % ( G ) of mouse p16-3MR VSMCs treated increasing concentrations of Doxorubicin for 1 day followed by 7 days recovery vs. vehicle control. ( I – K ) qPCR for Lamin B1, IL6, p16, or p21 mRNA expression for cells treated in ( F ). ( L ) Western blot of mouse cells as treated in ( F ) for Lamin B1, p16, or p21. n = 3–8 mouse VSMC isolates. Data are means (SD), one-way ANOVA with correction for multiple comparisons ( A ) or unpaired Student’s t -test vs. Control 1d ( B – D ) or vs. Vehicle (Dox 0 nM) ( F – K ).

Article Snippet: Sections were incubated with either primary antibodies: p16 (20 μg rabbit polyclonal, ProSci 4211), Smooth muscle cell α-actin-cy 3 -conjugated (1:1000 mouse monoclonal, Sigma-Aldrich C6198), CD68 (1:100 mouse monoclonal, Thermo 14-0689-82) or isotype control antibodies: rabbit monoclonal IgG isotype control (Abcam ab172730), mouse monoclonal IgG isotype control (Abcam ab37355) diluted in 3% BSA for 1 h at room temperature.

Techniques: Cell Culture, Control, Western Blot, Expressing

Journal: Cardiovascular Research

Article Title: Efficacy and limitations of senolysis in atherosclerosis

doi: 10.1093/cvr/cvab208

Figure Lengend Snippet: p16/Cdkn2a is detected in VSMCs in mouse atherosclerotic plaques. ( A and B ) UMAP plots showing scRNA-seq profiles of unsorted aortic cells from Myh11Cre ERt2+ /Confetti + mice ( A ), or sorted Confetti + VSMCs from atherosclerotic plaque and media of fat-fed Myh11Cre ERt2 /Confetti + /ApoE −/− mice ( B ). Log-transformed expression levels of Myh11 and p16/Cdkn2a are shown alongside e-Cadherin/Cdh5 and Pdgfrα ( A ) or Cd68 and Ly6a/Sca1 ( B ) using a scale from white to dark red. Insets show high power regions of clusters 6, 8, and 9 and expression of p16 in ( B ). Feature plots show log-normalized expression levels. ( C ) % cells in each cluster with detectable expression of p16/cdkn2a after 14 weeks or 18 weeks or high-fat feeding, or combined.

Article Snippet: Sections were incubated with either primary antibodies: p16 (20 μg rabbit polyclonal, ProSci 4211), Smooth muscle cell α-actin-cy 3 -conjugated (1:1000 mouse monoclonal, Sigma-Aldrich C6198), CD68 (1:100 mouse monoclonal, Thermo 14-0689-82) or isotype control antibodies: rabbit monoclonal IgG isotype control (Abcam ab172730), mouse monoclonal IgG isotype control (Abcam ab37355) diluted in 3% BSA for 1 h at room temperature.

Techniques: Transformation Assay, Expressing

Journal: Cardiovascular Research

Article Title: Efficacy and limitations of senolysis in atherosclerosis

doi: 10.1093/cvr/cvab208

Figure Lengend Snippet: GCV treatment of p16-3MR mice does not affect atherosclerosis, but induces inflammation. ( A ) Aortic root plaques in ApoE→ApoE, p16→ApoE, ApoE→p16, or p16→p16 mice + GCV, or p16→p16 mice + saline, stained with Masson’s trichrome, TUNEL, or Mac3. Scale bar = 300 µm. High power inset shows apoptotic cell and nuclear debris from outlined area. ( B ) Plaque area for mice in ( A ). ( C and D ) Number of TUNEL + cells/aortic root plaque ( C ) or %Mac3 + cells ( D ) for mice in ( A ). ( E – G ) Relative mRNA expression for p16, IL18, or TNFα in experimental mice. Data are means (SD) n = 5–10 mice. One-way ANOVA with correction for multiple comparisons ( B – D ) or Kruskal–Wallis H test followed by Dunn’s multiple comparisons test ( E – G ).

Article Snippet: Sections were incubated with either primary antibodies: p16 (20 μg rabbit polyclonal, ProSci 4211), Smooth muscle cell α-actin-cy 3 -conjugated (1:1000 mouse monoclonal, Sigma-Aldrich C6198), CD68 (1:100 mouse monoclonal, Thermo 14-0689-82) or isotype control antibodies: rabbit monoclonal IgG isotype control (Abcam ab172730), mouse monoclonal IgG isotype control (Abcam ab37355) diluted in 3% BSA for 1 h at room temperature.

Techniques: Saline, Staining, TUNEL Assay, Expressing

Journal: Cardiovascular Research

Article Title: Efficacy and limitations of senolysis in atherosclerosis

doi: 10.1093/cvr/cvab208

Figure Lengend Snippet: ABT-263 (Navitoclax) selectively reduces senescent VSMCs. ( A and B ) Photomicrographs ( A ) or quantification ( B ) of mouse VSMCs stained for SAβG, as replicating control cells or after dox1 + 7 days treatment, or each group ±1 µM ABT-263 treatment for 48 h. ( C ) Western blot and quantification for p16 in cells treated in ( A and B ). ( D ) Fold change in mRNA expression compared with control replicating cells for p16 and a range of SASP cytokines against the housekeeping gene HMBS. Data are means (SD), n = 4–5. Unpaired Student’s t -test. ( E ) Mouse macrophages cultured for 28 days, then treated with 1 µM ABT-263 for 48 h and stained for SAβG. Data are means (SD), n = 3. Unpaired Student’s t -test.

Article Snippet: Sections were incubated with either primary antibodies: p16 (20 μg rabbit polyclonal, ProSci 4211), Smooth muscle cell α-actin-cy 3 -conjugated (1:1000 mouse monoclonal, Sigma-Aldrich C6198), CD68 (1:100 mouse monoclonal, Thermo 14-0689-82) or isotype control antibodies: rabbit monoclonal IgG isotype control (Abcam ab172730), mouse monoclonal IgG isotype control (Abcam ab37355) diluted in 3% BSA for 1 h at room temperature.

Techniques: Staining, Control, Western Blot, Expressing, Cell Culture

Journal: Cardiovascular Research

Article Title: Efficacy and limitations of senolysis in atherosclerosis

doi: 10.1093/cvr/cvab208

Figure Lengend Snippet: ABT-263 reduces atherosclerosis, but not local SASP cytokine expression. ( A ) ORO staining of mouse descending aorta treated with control (vehicle) or ABT-263, and quantification of %ORO area ( n = 11–14). Scale bar = 3 mm. ( B ) Masson’s trichrome histochemistry of aortic root atherosclerotic plaque from mice treated in ( A ). Panels below show high power view of outlined area. Arrow shows necrotic core. Scale bar = 200 µm. ( C – E ) Aortic root plaque area/total area ( C ) Cap area ( D ), or Core area ( E ) for mice in ( A ). n = 11–13. ( F ) qPCR for relative expression of p16 or SASP cytokines in aortic arches of experimental mice against the housekeeping gene HMBS ( n = 7). Data are means (SD), n = 10. Unpaired Student’s t -test ( A, C – E ) or Mann–Whitney U test ( F ).

Article Snippet: Sections were incubated with either primary antibodies: p16 (20 μg rabbit polyclonal, ProSci 4211), Smooth muscle cell α-actin-cy 3 -conjugated (1:1000 mouse monoclonal, Sigma-Aldrich C6198), CD68 (1:100 mouse monoclonal, Thermo 14-0689-82) or isotype control antibodies: rabbit monoclonal IgG isotype control (Abcam ab172730), mouse monoclonal IgG isotype control (Abcam ab37355) diluted in 3% BSA for 1 h at room temperature.

Techniques: Expressing, Staining, Control, MANN-WHITNEY