p21  (Panlab)

Journal: Aging Cell

Article Title: High‐Fat Diet–Induced Metabolic Stress Durably Reprograms the Liver Regenerative Compartment Toward a Senescence‐Like State

doi: 10.1111/acel.70769

Figure Lengend Snippet: HFD induces aging‐like hepatic dysfunction under chronic metabolic stress. (A) Experimental design. (B) Body weight and caloric intake ( n = 41–44/group). (C) Liver and white adipose tissue (WAT) weights ( n = 9–11/group). (D) Serum total cholesterol (T‐Cho), HDL cholesterol (HDL‐C), non‐HDL cholesterol (non‐HDL‐C), and triglycerides (TG) ( n = 11/group). (E) Representative hematoxylin–eosin (HE) and Oil Red O staining of liver sections. Scale bar, 50 μm. (F) Hepatic TG content and lipid peroxidation (LPO) levels ( n = 6/group). (G) Hepatic inflammatory cytokine levels (IL‐1β, IL‐6, and TNF‐α) ( n = 5/group). (H) Representative images of SA‐β‐gal staining and immunostaining for p16, p21, γH2AX, and Lamin B1 in liver sections. Arrows indicate positive cells. Scale bars, 100 μm. (I) Quantification of the SA‐β‐gal‐positive area, the numbers of p16‐, p21‐, and γH2AX‐positive cells, and Lamin B1 fluorescence intensity ( n = 4–5/group). Data are presented as mean ± SD. Body weight and caloric intake were analyzed using Bonferroni's multiple comparison test. All other data were analyzed using Student's t ‐test. * p < 0.05; ** p < 0.01.

Article Snippet: For immunohistochemistry, sections were incubated with mouse anti‐p16 (1:400; Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti‐p21 (1:2000; Proteintech, Rosemont, IL, USA), mouse anti‐γH2AX (1:2000; Santa Cruz Biotechnology), or rabbit anti‐Ki67 (1:800; Abcam, Cambridge, UK) followed by Histofine Simple Stain MAX‐PO (MULTI) and DAB visualization.

Techniques: Tissue, Staining, Immunostaining, Fluorescence, Comparison

Journal: Aging Cell

Article Title: High‐Fat Diet–Induced Metabolic Stress Durably Reprograms the Liver Regenerative Compartment Toward a Senescence‐Like State

doi: 10.1111/acel.70769

Figure Lengend Snippet: HFD impairs regenerative responses and enhances senescence‐associated changes after CCl 4 ‐induced liver injury. (A) CCl 4 ‐induced liver injury and regeneration model. In this injury context, facultative regenerative programs involving liver stem/progenitor cells have been reported to contribute, at least in part, to epithelial repair. (B) Eight weeks of dietary feeding followed by two CCl 4 injections within 1 week (1 mL/kg body weight diluted 1:1 in olive oil). Regenerating livers were assessed 2 weeks after the final injection. (C) Representative staining images and quantification of the percentage of Ki67‐positive cells in regenerating livers (% nuclei; n = 3–6 per group). (D) Representative staining images of p16, p21, and γH2AX in regenerating liver tissue. Arrows indicate positive cells. Scale bar: 100 μm. (E) Quantification of the percentage of senescence marker‐positive cells in regenerating livers (% nuclei; n = 3–6/group). (F) Temporal changes in the percentages of p16‐, p21‐, and γH2AX‐positive cells before and after CCl 4 injury ( n = 3–6/group). (G) p16‐ and γH2AX‐positive cells among Ki67‐positive cells in regenerating livers ( n = 3–6/group). Data are presented as mean ± SD. * p < 0.05; ** p < 0.01, Student's t ‐test.

Article Snippet: For immunohistochemistry, sections were incubated with mouse anti‐p16 (1:400; Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti‐p21 (1:2000; Proteintech, Rosemont, IL, USA), mouse anti‐γH2AX (1:2000; Santa Cruz Biotechnology), or rabbit anti‐Ki67 (1:800; Abcam, Cambridge, UK) followed by Histofine Simple Stain MAX‐PO (MULTI) and DAB visualization.

Techniques: Injection, Staining, Tissue, Marker, Stem Cell

Journal: Aging Cell

Article Title: High‐Fat Diet–Induced Metabolic Stress Durably Reprograms the Liver Regenerative Compartment Toward a Senescence‐Like State

doi: 10.1111/acel.70769

Figure Lengend Snippet: Liver organoids from HFD‐fed rats exhibit reduced formation capacity and a persistent senescence‐associated phenotype. (A) Liver tissues from control and HFD‐fed rats were dissociated, and regenerative cell–enriched fractions were cultured in Matrigel domes to generate organoids. (B) Representative images of primary organoids. Scale bar, 500 μm. (C) Organoid size distribution and diameter ( n = 6–10/group). (D) Organoids were mechanically disrupted and replated at each passage to assess self‐renewal capacity. (E) Representative images of organoids at Passages 1 (P1), 5, 8, 10, and 13. Scale bar, 200 μm. (F) Organoid formation efficiency during serial passaging. The formation rate is expressed as a percentage of the number of P1 organoids and is normalized to 100% ( n = 6–10/group). (G) Kaplan–Meier survival analysis of organoid formation. Loss of organoid formation was defined as the absence of organoids measuring > 100 μm in diameter ( n = 6–10/group). (H) Representative images of SA‐β‐gal staining and immunofluorescence staining for p16, p21, γH2AX, Ki67, and Lamin B1 in organoid sections at P1–3. DAPI (blue) labels nuclei. Green fluorescence indicates p16, γH2AX, and Lamin B1, whereas red fluorescence indicates p21 and Ki67. Scale bar, 50 μm. (I) Quantification of the SA‐β‐gal‐positive area, the numbers of p16‐, p21‐, γH2AX‐, and Ki67‐positive cells, and Lamin B1 fluorescence intensity at each passage ( n = 4–6/group). (J) Passage‐dependent changes in each senescence‐associated marker ( n = 4–6/group). Each animal‐derived organoid culture was considered one biological replicate. Formation efficiency is presented as mean ± SE; all other data are presented as mean ± SD. Survival curves were analyzed using the Kaplan–Meier method, and statistical significance was determined using the log‐rank test. All other data were analyzed using Student's t ‐test. * p < 0.05; ** p < 0.01.

Article Snippet: For immunohistochemistry, sections were incubated with mouse anti‐p16 (1:400; Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti‐p21 (1:2000; Proteintech, Rosemont, IL, USA), mouse anti‐γH2AX (1:2000; Santa Cruz Biotechnology), or rabbit anti‐Ki67 (1:800; Abcam, Cambridge, UK) followed by Histofine Simple Stain MAX‐PO (MULTI) and DAB visualization.

Techniques: Organoids, Control, Cell Culture, Passaging, Analysis, Staining, Immunofluorescence, Fluorescence, Marker, Derivative Assay

Journal: bioRxiv

Article Title: p53 activity links chromosomal instability to pro-migratory extracellular vesicles

doi: 10.64898/2026.10.06.757210

Figure Lengend Snippet: a. Schematic overview of the experimental design . b. Quantification of mitotic errors in NIH/3T3 cells treated with DMSO, reversine (REV; 75 nM), or doxorubicin (DOXO; 250 nM). Mps1 inhibition by REV compromised spindle-assembly-checkpoint control and increased chromosome-segregation errors. No mitotic events were observed in the DOXO-treated group during imaging; therefore, the frequency of mitotic errors could not be calculated for this condition. c. Single-cell whole-genome sequencing (scWGS) analysis of NIH/3T3 cells treated with DMSO, REV, or DOXO, showing treatment-associated chromosome copy-number alterations. Each row represents one cell, and colors indicate chromosome copy-number states. d. Quantitative PCR analysis of the relative expression of Trp53 , Cdkn1a , Il6, and Lmnb1 in NIH/3T3 cells treated with DMSO, REV, or DOXO. e. Quantification of the percentage of EdU-positive cells and SA-β-galactosidase activity following DMSO, REV, or DOXO treatment. SA-β-galactosidase activity is expressed as integrated density per cell. f. Schematic of the p21–RFP reporter construct, in which TurboRFP expression is driven by the murine Cdkn1a promoter containing p53-binding sites. g. Representative flow-cytometry analysis of RFP expression in NIH/3T3 p21–RFP reporter cells treated with DMSO or REV (75 nM, 96 h), showing reporter-defined p21-RFP HIGH and p21-RFP LOW populations. h. Validation of reporter dependence on p53 using two independently generated Trp53 -knockout NIH/3T3 reporter clones analyzed by flow cytometry. i. Longitudinal characterization of REV-induced p21-RFP HIGH and p21-RFP LOW populations after sorting. RFP fluorescence and EdU incorporation following a 24h pulse was measured on days 1–6 (n = 2 each). p21 protein abundance was assessed by western blot densitometry (n = 1), and SA-β-galactosidase activity was quantified (n = 3) on days 2, 4, and 6. n denotes independent experiments. Data in panels d and e are presented as mean ± SD. SA-β-galactosidase data in panel i are presented as mean ± SD; RFP and EdU plots show the mean and individual values from two independent experiments. Time-course data are presented descriptively without statistical hypothesis testing. Statistical significance was determined using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test for panels ( d ) and ( e ) (n = 4 independent experiments for panel d ; n = 3 independent experiments for panel e ). Abbreviations: DMSO, vehicle control; REV, reversine; DOXO, doxorubicin. Statistical significance is indicated as follows: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: NIH/3T3 cells were transfected with the p21 promoter–driven TurboRFP reporter plasmid (pTurboRFP-PRL, Addgene #52432) using Lipofectamine 3000 (Thermo Fisher).

Techniques: Inhibition, Control, Imaging, Single Cell, Whole-Genome Sequencing, Analysis, Real-time Polymerase Chain Reaction, Expressing, Activity Assay, Construct, Binding Assay, Flow Cytometry, Biomarker Discovery, Generated, Knock-Out, Clone Assay, Fluorescence, EdU Assay, Quantitative Proteomics, Western Blot, Genomic Sequencing

Journal: bioRxiv

Article Title: p53 activity links chromosomal instability to pro-migratory extracellular vesicles

doi: 10.64898/2026.10.06.757210

Figure Lengend Snippet: a. Principal component analysis (PCA) of transcriptomes across all 13 experimental groups, including unsorted controls and longitudinally followed reporter-sorted populations. b. K-means clustering analysis of differentially expressed genes (DEGs) across all treatment groups and time points, identifying six major transcriptional clusters. c. Hallmark pathway enrichment analysis of the six transcriptional clusters identified in panel b. d. Heatmap showing genes upregulated in cluster 5 across treatments and time points, grouped by functional categories. e. Hallmark pathway signature analyses comparing REV-induced p21-RFPLOW cells (left) and p21-RFPHIGH cells (right) with DMSO controls at day 6 after sorting. f. Volcano plot showing differentially expressed genes between REV-induced p21-RFPHIGH and p21-RFPLOW cells at day 6 after sorting. Positive log₂ fold changes indicate higher expression in p21-RFPHIGH cells, whereas negative values indicate higher expression in p21-RFPLOW cells. Selected genes are labeled.

Article Snippet: NIH/3T3 cells were transfected with the p21 promoter–driven TurboRFP reporter plasmid (pTurboRFP-PRL, Addgene #52432) using Lipofectamine 3000 (Thermo Fisher).

Techniques: Analysis, Functional Assay, Expressing, Labeling

Journal: bioRxiv

Article Title: p53 activity links chromosomal instability to pro-migratory extracellular vesicles

doi: 10.64898/2026.10.06.757210

Figure Lengend Snippet: a. Experimental overview of EV isolation and mass spectrometry–based proteomic analysis using unsorted NIH/3T3 p21-RFP reporter cells treated with DMSO, REV, or DOXO. b. Particle-size distributions of EV preparations isolated from conditioned media of DMSO-, REV-, or DOXO-treated cells, measured by nanoparticle tracking analysis (NTA). Lines and shaded areas represent the mean and SD, respectively. c. EV particle numbers measured by NTA and normalized to viable donor-cell number at harvest, expressed per 10⁶ viable donor cells. d. Principal component analysis (PCA) of EV proteomes from DMSO-, reversine-, and doxorubicin-treated groups. e. K-means clustering of EV-associated proteins across treatment groups, identifying five clusters, with corresponding Hallmark and KEGG pathway enrichment analyses. f. GOBP enrichment analysis comparing EVs isolated from reversine- and doxorubicin-treated cells to EVs isolated from DMSO controls. Shown are the mean ± SD values. Statistical significance was determined using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test for panels (c) (n = 3 independent experiments for panels c). Abbreviations: DMSO, vehicle control; REV, reversine; DOXO, doxorubicin. Statistical significance is indicated as follows: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: NIH/3T3 cells were transfected with the p21 promoter–driven TurboRFP reporter plasmid (pTurboRFP-PRL, Addgene #52432) using Lipofectamine 3000 (Thermo Fisher).

Techniques: Isolation, Mass Spectrometry, Analysis, Control

Journal: bioRxiv

Article Title: p53 activity links chromosomal instability to pro-migratory extracellular vesicles

doi: 10.64898/2026.10.06.757210

Figure Lengend Snippet: a. Principal component analysis (PCA) of EV proteomes derived from sorted cell populations and control. b. K-means clustering of EV proteomes from all experimental groups. c, d . Hallmark ( c ) and KEGG ( d ) pathway enrichment analyses of the two protein clusters identified in panel b . e. K-means clustering of proteins significantly increased in day 6 REV-induced p21-RFPHIGH-derived EVs compared with DMSO-derived EVs, identifying three clusters whose abundance patterns are shown across all samples. f. Boxplots showing normalized protein intensities (“MS values,” log-transformed and median-normalized) in EVs isolated from reversine-treated p21-RFP HIGH cells over time, compared to proteins in EVs isolated from reversine-treated p21-RFP LOW cells and DMSO control cells. Colors indicate median z-scores corresponding to the heatmap (blue = downregulated, red = upregulated). g–h . GO Molecular Function enrichment analyses of proteins in clusters 2 ( g ) and 3 ( h ) identified in panel e , using all annotated genes/proteins in the analysis database as the background. Only terms meeting the significance criterion used in the enrichment analysis are shown. Bar lengths indicate −log₁₀(Q-value). Numbers beside the bars indicate the number of proteins from the respective cluster associated with each term.

Article Snippet: NIH/3T3 cells were transfected with the p21 promoter–driven TurboRFP reporter plasmid (pTurboRFP-PRL, Addgene #52432) using Lipofectamine 3000 (Thermo Fisher).

Techniques: Analysis, Derivative Assay, Control, Transformation Assay, Isolation, Protein Binding

Journal: bioRxiv

Article Title: p53 activity links chromosomal instability to pro-migratory extracellular vesicles

doi: 10.64898/2026.10.06.757210

Figure Lengend Snippet: a. Volcano plot comparing EV protein abundance between day 6 REV-induced p21-RFP HIGH and p21-RFP LOW NIH/3T3 cells. Red and blue indicate significantly increased and decreased proteins, respectively, in p21-RFP HIGH -derived EVs; gray indicates proteins not meeting the significance thresholds. Selected proteins are labeled. b, c. KEGG pathway ( b ) and GO Molecular Function ( c ) enrichment analyses of differentially abundant EV proteins identified in the day 6 p21-RFP HIGH and p21-RFP LOW comparison. Bubble size indicates protein count, color represents −log₁₀(FDR), and the x-axis shows the gene ratio (%). d. Representative fluorescence images of 4T1 recipient cells incubated for 4 h with PKH67-labeled EV preparations from DMSO- or REV-treated NIH/3T3 cells. PBS and a 4 °C incubation condition were included as controls. PKH67 is shown in green, F-actin in red, and nuclei in blue. Scale bar: 50 μm. e. Experimental workflow for EV collection from REV-induced p21-RFP HIGH and p21-RFP LOW NIH/3T3 cells on day 6 after sorting and subsequent treatment of 4T1 recipient cells for scratch-wound assays. f, g. Representative images at 0 h (top) and 24 h (bottom) after scratching ( f ) and quantification of scratch-wound closure at 24 h ( g ). Recipient 4T1 cells were treated with EVs derived from REV-induced p21-RFP LOW or p21-RFP HIGH cells. Scale bar: 400 μm. Bars represent mean ± SD, and each point represents one independent experiment (n = 3). Statistical significance was determined using an unpaired two-tailed Student’s t-test for panel g . Abbreviations: DMSO, vehicle control; REV, reversine. Statistical significance is indicated as follows: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: NIH/3T3 cells were transfected with the p21 promoter–driven TurboRFP reporter plasmid (pTurboRFP-PRL, Addgene #52432) using Lipofectamine 3000 (Thermo Fisher).

Techniques: Quantitative Proteomics, Derivative Assay, Labeling, Comparison, Fluorescence, Incubation, Two Tailed Test, Control, Significance Assay, Protein Binding

Journal: bioRxiv

Article Title: p53 activity links chromosomal instability to pro-migratory extracellular vesicles

doi: 10.64898/2026.10.06.757210

Figure Lengend Snippet: Acute CIN induces heterogeneous p53–p21 responses, generating p21 HIGH and p21 LOW cell states with distinct recovery trajectories. p21 LOW cells re-enter the cell cycle earlier, whereas p21 HIGH cells maintain a more persistent arrest and progressively develop ECM-, adhesion-, and TGF-β-associated transcriptional programs. This state is accompanied by remodeling of EV protein cargo and the release of EVs enriched in ECM-, adhesion-, and cytoskeleton-associated proteins. Compared with EVs from p21-RFP LOW cells, EVs released by p21 HIGH cells enhance scratch-wound closure in recipient tumor cells, linking persistent p21 activation to a distinct, migration-associated paracrine program.

Article Snippet: NIH/3T3 cells were transfected with the p21 promoter–driven TurboRFP reporter plasmid (pTurboRFP-PRL, Addgene #52432) using Lipofectamine 3000 (Thermo Fisher).

Techniques: Activation Assay, Migration