p21  (Santa Cruz Biotechnology)

Journal: Bioactive Materials

Article Title: Gynostemma pentaphyllum -derived extracellular vesicles alleviate skin aging by destabilizing STING

doi: 10.1016/j.bioactmat.2026.03.010

Figure Lengend Snippet: GPEVs improve d UVB irradiation-induced senescence of HEKa cells in vitro. (A) The SA-β-gal staining of UVB-irradiated HEKa (6 mJ/cm 2 ) cells subjected to treatments with a range of GPEVs concentrations (1 × 10 8 , 2 × 10 8 , 4 × 10 8 particles/ml) and NAC (1 mM). Scale bar: 50 μm. (B) The Quantification of SA-β-gal staining in A. (C) Representative images of EdU staining in UVB-irradiated HEKa cells following treatment with NAC and varying concentrations of GPEVs. Scale bar: 50 μm. (D) Quantification of EdU-positive cells in C. (E) The protein levels of p16, p21 and p53 in HEKa cells exposed to UVB irradiation and GPEVs treatment were assessed using Western blot analysis. (F) The quantification of E was performed using Image J software. (G) The relative mRNA levels of the senescence-associated secretory phenotype in irradiated HEKa cells treated with or without GPEVs were measured by RT-qPCR. All the experiments were repeated at least three times. Data are represented as means ± SD. Statistical analysis was performed using one-way ANOVA followed by Bonferroni post hoc test. Not significant (ns), ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: The primary antibodies used included γ-H2AX and CTCF (1:1,000, Cell Signaling), p21 and STING (diluted 1:1,000, Cell Signaling).

Techniques: Irradiation, In Vitro, Staining, Western Blot, Software, Quantitative RT-PCR

Journal: Bioactive Materials

Article Title: Gynostemma pentaphyllum -derived extracellular vesicles alleviate skin aging by destabilizing STING

doi: 10.1016/j.bioactmat.2026.03.010

Figure Lengend Snippet: The application of GPEVs effectively mitigate d UVB-induced skin aging in Balb/C mice. (A) Representative images of Masson's trichrome staining of mice skin from each group. Collagen fibers are stained blue. Scale bar: 200 μm. (B) Quantitative analysis of collagen fibers in Masson's trichrome stained skin tissue sections by Image J software. (C and D) The levels of p21 in mice skin were detected by immunofluorescent staining and quantified using Image J software. (E) The protein levels of aging-related proteins in skin tissues were determined by Western Blots. (F) The quantification of E was conducted using Image J software. (G) The mRNA levels of senescence-associated secretory phenotype in skin tissues were detected by RT-qPCR. Data are represented as means ± SD. Statistical analysis was performed using One-way ANOVA followed by Bonferroni post hoc test. Not significant (ns), ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: The primary antibodies used included γ-H2AX and CTCF (1:1,000, Cell Signaling), p21 and STING (diluted 1:1,000, Cell Signaling).

Techniques: Staining, Software, Western Blot, Quantitative RT-PCR

Journal: Bioactive Materials

Article Title: Microenvironment-educated MSC-EVs loaded injectable smart hydrogel for targeting senescent nucleus pulposus cells and inhibiting ferroptosis against intervertebral disc degeneration

doi: 10.1016/j.bioactmat.2026.02.030

Figure Lengend Snippet: Senescent Microenvironment-Educated Mesenchymal Stem Cells Release High-Affinity Senescent NPC Domesticated Extracellular Vesicles. (A) Schematic diagram of the experimental setup for educating MSCs with SASP-CM to generate D-EVs versus N-EVs. (B) Confocal microscopy images showing different EVs internalization by senescent NPCs after 12 h in vitro. (C) Flow cytometry and quantification analysis of different EVs uptake by senescent NPCs. (D) In vivo validation of the senescent niche. Representative fluorescence images following injection of senescence-tracer (Red). (E) In vivo PKH26-labeled D-EVs tracking. (F) Representative SA-β-Gal images and quantification of MSCs treated with SASP-CM or not. (G) Gene Ontology (GO) analysis confirming enrichment of external encapsulating structure organization and cytokine production in Biological Process (BP) categories. (H) Heatmap indicating gene expression associated with EVs biogenesis within D-MSCs and N-MSCs. (I) Heatmap indicating gene expression associated with cytokine production within D-MSCs and N-MSCs. (J and L) Gene Ontology (GO) analysis confirming enrichment of terms related to vesicle organization and transport in the Cellular Component (CC) categories. (K) Western blot analysis confirmed core senescence markers p16 and p21 and DNA damage marker γ-H2AX in N-MSC and D-MSC. (M) Western blot analysis confirmed the expression of CD9, CD63, TSG101, Calnexin, and GM130 in MSC-EVs, N-EVs, or D-EVs. (N) TEM images showing the morphology and size of MSC-derived EVs, N-EVs, and D-EVs. (O) NTA shows size distribution in MSC-EVs, N-EVs, or D-EVs. The data were presented as mean ± SD. n = 3, ns, not significant; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: After blocked with 5% non-fat milk for 2 h at room temperature, the membranes were incubated with primary antibodies against GAPDH (1:5000, 104941-AP, Proteintech), TSG101 (1:1000, DF8427, Affinity), CD9 (1:1000, AF5139, Affinity), CD63 (1:2000, 25682-1-AP, Proteintech), Calnexin (1:5000, 10427-2-AP, Proteintech), GM130 (1:20000, 11308-1-AP, Proteintech), CXCR3 (1:5000, 26756-1-AP, Proteintech), CXCL10 (1:2000, 10937-1-AP, Proteintech), MMP3 (1:2000, 17873-1-AP, Proteintech), ADAMTS5 (DF13268, Affinity), P16 (AF5484, Affinity), P21 (10355-1-AP, Proteintech), GPX4 (1:1000, 381958, Zen-bio), SLC7A11 (1:1000, 26864-1-AP, Proteintech), ACSL4 (1:5000, 22401-1-AP, Proteintech) and Tubulin (1:10000, T40103 , Abmart) overnight at 4 °C.

Techniques: Confocal Microscopy, In Vitro, Flow Cytometry, In Vivo, Biomarker Discovery, Fluorescence, Injection, Labeling, Gene Expression, Western Blot, Marker, Expressing, Derivative Assay

Journal: Bioactive Materials

Article Title: Microenvironment-educated MSC-EVs loaded injectable smart hydrogel for targeting senescent nucleus pulposus cells and inhibiting ferroptosis against intervertebral disc degeneration

doi: 10.1016/j.bioactmat.2026.02.030

Figure Lengend Snippet: D-EVs Alleviate Cellular Senescence and Restore ECM anabolic/catabolic metabolism in Senescent NPCs. (A) The CCK8 assay was used to determine D-EVs concentrations on cell viability. (B) Flow cytometry analysis of proliferative capacity in the above group, and (C) quantitative analysis. (D) Representative ROS images of senescent NPCs treated with N-EVs, D-EVs, or D-EVs + GW4869. (E) Representative SA-β-Gal images of senescent NPCs treated with N-EVs, D-EVs, or D-EVs + GW4869, and (F) quantitative analysis. (G) Confocal analysis of γ-H2A with IF staining depicting DNA damage in the control, TBHP, N-Evs, or D-EVs group. (H) WB analysis of ECM metabolism–related and aging-related proteins in NPCs following treatment with Control, TBHP, N-Evs, or D-EVs. (I) Western blot analysis of p53, p21, and p16 in senescent NPCs treated with D-EVs, D-CM, or D-CM EV-dep . (J) Confocal analysis of COL2 with IF staining in the control, TBHP, N-EVs, or D-EVs group. The data were presented as mean ± SD. n = 3, ns, not significant; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: After blocked with 5% non-fat milk for 2 h at room temperature, the membranes were incubated with primary antibodies against GAPDH (1:5000, 104941-AP, Proteintech), TSG101 (1:1000, DF8427, Affinity), CD9 (1:1000, AF5139, Affinity), CD63 (1:2000, 25682-1-AP, Proteintech), Calnexin (1:5000, 10427-2-AP, Proteintech), GM130 (1:20000, 11308-1-AP, Proteintech), CXCR3 (1:5000, 26756-1-AP, Proteintech), CXCL10 (1:2000, 10937-1-AP, Proteintech), MMP3 (1:2000, 17873-1-AP, Proteintech), ADAMTS5 (DF13268, Affinity), P16 (AF5484, Affinity), P21 (10355-1-AP, Proteintech), GPX4 (1:1000, 381958, Zen-bio), SLC7A11 (1:1000, 26864-1-AP, Proteintech), ACSL4 (1:5000, 22401-1-AP, Proteintech) and Tubulin (1:10000, T40103 , Abmart) overnight at 4 °C.

Techniques: CCK-8 Assay, Flow Cytometry, Staining, Control, Western Blot

Journal: Bioactive Materials

Article Title: Microenvironment-educated MSC-EVs loaded injectable smart hydrogel for targeting senescent nucleus pulposus cells and inhibiting ferroptosis against intervertebral disc degeneration

doi: 10.1016/j.bioactmat.2026.02.030

Figure Lengend Snippet: D-EVs Counteract NPC Senescence by Suppressing Ferroptosis. (A) KEGG pathway analysis of DEGs in senescent NPCs following treatment with D-EVs or not. (B-C) GSEA plots showing significant enrichment of ferroptosis and cell cycle in senescent NPCs. (D-E) Heatmap quantification of key genes involved in ferroptosis and cell cycle. (F) Western blot analysis of key ferroptosis (GPX4, SLC7A11, ACSL4) and senescence (p21, P16) markers in NPCs following treatment with different experimental conditions. (G) Representative images of C11-BODIPY 581/591 staining to detect lipid peroxidation (green) in the control, TBHP, Era, Era + Fer-1, or TBHP + Fer-1 groups. (H-I) Quantitative assessment of malondialdehyde (MDA) levels (H) and glutathione (GSH) levels (I) in the control, TBHP, N-EVs, D-EVs, or D-EVs + Era groups. (J) Western blot analysis of key ferroptosis (GPX4, SLC7A11, ACSL4) and senescence (p21, P16) markers in NPCs following treatment with PBS, N-EVs, D-EVs, or D-EVs + Era. (K) Confocal analysis of GPX4 with IF staining in the control, TBHP, N-Evs, D-EVs, and D-EVs + Era group. (L) Flow cytometry analysis of cell cycle distribution in the above experimental conditions. Statistical comparisons were performed between the experimental group and the TBHP-induced group. The data were presented as mean ± SD. n = 3, ns, not significant; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: After blocked with 5% non-fat milk for 2 h at room temperature, the membranes were incubated with primary antibodies against GAPDH (1:5000, 104941-AP, Proteintech), TSG101 (1:1000, DF8427, Affinity), CD9 (1:1000, AF5139, Affinity), CD63 (1:2000, 25682-1-AP, Proteintech), Calnexin (1:5000, 10427-2-AP, Proteintech), GM130 (1:20000, 11308-1-AP, Proteintech), CXCR3 (1:5000, 26756-1-AP, Proteintech), CXCL10 (1:2000, 10937-1-AP, Proteintech), MMP3 (1:2000, 17873-1-AP, Proteintech), ADAMTS5 (DF13268, Affinity), P16 (AF5484, Affinity), P21 (10355-1-AP, Proteintech), GPX4 (1:1000, 381958, Zen-bio), SLC7A11 (1:1000, 26864-1-AP, Proteintech), ACSL4 (1:5000, 22401-1-AP, Proteintech) and Tubulin (1:10000, T40103 , Abmart) overnight at 4 °C.

Techniques: Western Blot, Staining, Control, Flow Cytometry

Journal: Bioactive Materials

Article Title: Microenvironment-educated MSC-EVs loaded injectable smart hydrogel for targeting senescent nucleus pulposus cells and inhibiting ferroptosis against intervertebral disc degeneration

doi: 10.1016/j.bioactmat.2026.02.030

Figure Lengend Snippet: D-EVs Deliver GPX4 to Inhibit Ferroptosis in Senescent NPCs. (A) Representative Senescent-Tracker images of NPCs treated with N-EVs, D-EVs, Era, and D-Evs sh-CXCL10 . (B) Volcano plot of transcriptomic data comparing D-MSC and N-MSC. (C) KEGG pathway analysis of DEGs in D-MSCs versus N-MSCs. (D) Volcano plot of proteomic data comparing D-EVs and N-EVs. (E) KEGG pathway analysis of transcriptomic and proteomic data integration. (F) A Venn diagram illustrating the intersection of genes from the D-MSC transcriptome, the D-EVs proteome, and the ferroptosis-related gene set. (G) Bar graph showing the relative expression levels of core overlapping genes identified in (F). (H) MS analysis revealed that GPX4 is enriched in the D-EVs proteome. (I) Western blot analysis confirming GPX4 protein in D-EVs and N-EVs. (J) Western blot analysis of key senescence (p21, P16) markers in NPCs following treatment with PBS or N-EVs with CXCL10 or GPX4 knockout. (K) Representative images of EdU depicting cell proliferation ability in the control, TBHP, D-EVs, D-EVs sh-CXCL10 , D-EVs sh-GPX4 , and D-EVs sh-CXCL10+GPX4 groups. (L-M) Confocal images showing GPX4 delivery from different EVs to senescent NPCs at 12h and 24h co-culture, and (N) colocalization analysis. The data were presented as mean ± SD. n = 3, ns, not significant; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: After blocked with 5% non-fat milk for 2 h at room temperature, the membranes were incubated with primary antibodies against GAPDH (1:5000, 104941-AP, Proteintech), TSG101 (1:1000, DF8427, Affinity), CD9 (1:1000, AF5139, Affinity), CD63 (1:2000, 25682-1-AP, Proteintech), Calnexin (1:5000, 10427-2-AP, Proteintech), GM130 (1:20000, 11308-1-AP, Proteintech), CXCR3 (1:5000, 26756-1-AP, Proteintech), CXCL10 (1:2000, 10937-1-AP, Proteintech), MMP3 (1:2000, 17873-1-AP, Proteintech), ADAMTS5 (DF13268, Affinity), P16 (AF5484, Affinity), P21 (10355-1-AP, Proteintech), GPX4 (1:1000, 381958, Zen-bio), SLC7A11 (1:1000, 26864-1-AP, Proteintech), ACSL4 (1:5000, 22401-1-AP, Proteintech) and Tubulin (1:10000, T40103 , Abmart) overnight at 4 °C.

Techniques: Expressing, Western Blot, Knock-Out, Control, Co-Culture Assay

Journal: bioRxiv

Article Title: Developmental senescence orchestrates hyaloid vessel regression in the postnatal eye

doi: 10.64898/2026.05.12.724389

Figure Lengend Snippet: a -Transcriptome analyses from P5 hyaloid bulk-RNA sequencing data from R.A. Lang group compared to P5 retina pseudo bulk single-cell RNA sequencing data from S. Blackshaw group. Enrichment analysis was performed on selected mouse gene sets by Gene Set Variation Analysis (GSVA) and coefficient comparison was performed using limma with the difference in GSVA enrichment score (contrast fit coefficient) displayed as bar plot. Blue arrows point senescence-related pathways. b- Quantitative reverse transcription polymerase chain reaction (qRT-PCR) of SASP-related genes ( Tgf-β1, Il1β, Il6, Serpine1 ) comparing between hyaloids and retina at P0, P4 and P8. β-Actin was used as reference gene. Data are presented as fold change compared to retina at each developmental stage (n=3). Bar graphs are means ± SEM. Represented P values are **<0.01, ***<0.001 and **** < 0.0001 from two-tailed parametric unpaired t -test. c - Bar charts of qRT-PCR from SASP-related genes ( Tgf-β1, Il1β, Il6, Serpine1, Vegf-a ) and d- cell cycle arrest markers ( Cdkn1a, Tp53 ) in hyaloids at P0, P4 and P8. Lmnb1 is used as a negative control. β-Actin was used as a reference gene. Data are presented as fold change compared to P0 ( n ≥3-6). Bar graphs are represented as means ± SEM. Represented P values are *<0.05, **<0.01, ***<0.001 and ****<0.0001 from ordinary one-way ANOVA test followed by Dunnett’s multiple comparisons for Tgf-β1, Il1β, Il6, Serpine1, Vegf-a and Cdkn1a and Kruskal-Wallis test followed by Dunn’s multiple comparisons for Tp53 . e - Immunoblots of hyaloid cell lysates from P0, P4 and P8 for senescence markers (P21, TP53, PAI-1). β-ACTIN was used as a loading control (n = 3). f - Representative confocal immunofluorescence staining of P21 (green) and g - PAI-1 (green) of flat-mounted hyaloid vessels at P0, P4 and P8. Blood vessels were stained with IB4 (red). Scale bars, 100 µm. Non-significant (ns).

Article Snippet: C57BL/6 wild-type and Cdkn1a (p21) ( Cdkn1a tm1Led /J, no. 016565) knock-out ( Cdkn1a -/- ) [ ] mice lines were obtained from The Jackson Laboratory.

Techniques: RNA Sequencing, Single Cell, Comparison, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Two Tailed Test, Negative Control, Western Blot, Control, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Developmental senescence orchestrates hyaloid vessel regression in the postnatal eye

doi: 10.64898/2026.05.12.724389

Figure Lengend Snippet: a- Uniform manifold approximation and projection (UMAP) of single-cell RNA sequencing from P4 hyaloid vessels representing the 9 hyaloid cell types identified by graph-based clustering of normalized RNA count. b- Heat map displaying log2 FC of VISION calculated enrichment scores across the 9 hyaloid cell populations for selected senescence- and c - SASP-related gene sets, show enrichment in immune, VSMCs and endothelial cell clusters. d - Dot plot illustrating the expression levels of SenMayo-related genes across the 9 hyaloid cell clusters. e- UMAP feature plot representation of Cdkn1a expression across the 9 hyaloid cell populations. f - Relative mRNA levels of Cdkn1a in P8 hyaloid vessels Cdkn1a +/+ versus Cdkn1a -/- measured by qRT-PCR (n = 3). β-Actin was used as reference gene. g- Representative IB4 and h- SA-β-Gal staining of flat-mounted P8 hyaloid vessels Cdkn1a +/+ (control) and Cdkn1a -/- (lacking Cdkn1a). Higher magnifications of vessels are shown. i - Quantification of total vessel length of P8 flat-mounted hyaloids Cdkn1a +/+ control versus Cdkn1a -/- . Each dot represents a flat-mounted hyaloid sample (n = 8-11). j- Quantification of SA-β-Gal staining per 100 µm of vessels (in percentage) of P8 Cdkn1a +/+ versus Cdkn1a -/- flat-mounted hyaloid. k- Representative IB4 staining of P8 retinal flat-mount Cdkn1a +/+ and Cdkn1a -/- and l - quantification of radial outgrowth of retinal vascular plexus (n = 3), showing delayed expansion of the superficial vascular plexus in Cdkn1a -/- mice. Data are presented as fold change normalized to Cdkn1a +/+ control ( f, j, l ) or as Arbitrary Unit (A.U.) ( i ). Data are represented as means ± SEM ( f, j, l ) or as individual values ( i ). Represented P values are * * < 0.01, *** < 0.001 and **** < 0.0001 from two-tailed parametric unpaired t -test. Scale bars, 500 µm ( g, h ) and 100 µm ( k ) [for higher magnification in g, h ].

Article Snippet: C57BL/6 wild-type and Cdkn1a (p21) ( Cdkn1a tm1Led /J, no. 016565) knock-out ( Cdkn1a -/- ) [ ] mice lines were obtained from The Jackson Laboratory.

Techniques: Single Cell, RNA Sequencing, Expressing, Quantitative RT-PCR, Staining, Control, Two Tailed Test