p21  (Novus Biologicals)

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: iScience

Article Title: Identification and validation of key PANoptosis-related genes via integrative machine learning and single-cell sequencing in AILI

doi: 10.1016/j.isci.2026.115183

Figure Lengend Snippet: Expression and functional exploration of the key genes in single-cell sequencing data (A) The UMAP plot shows the total sample composition, tissue sources, and cell subtypes. (B) The stacked graph shows the proportion of each type of cell in the control group and the AILI group. (C) Bubble plots of marker gene expression demonstrating the accuracy of the cell annotations. (D) Bubble chart showing the expression of Cdkn1a and Pdk1 in various cells in the control group. (E) Bubble chart showing the expression of Cdkn1a and Pdk1 in various cells in the AILI group. (F) Circle plot and heatmap showing the cell communication weights and numbers of all cell subtypes. (G–J) Receptor‒ligand communication weights between AILI and control samples.

Article Snippet: p21 Polyclonal antibody , Proteintech , Cat No.28248-1-AP; RRID: AB_2881097.

Techniques: Expressing, Functional Assay, Single Cell, Sequencing, Control, Marker, Gene Expression

Journal: iScience

Article Title: Identification and validation of key PANoptosis-related genes via integrative machine learning and single-cell sequencing in AILI

doi: 10.1016/j.isci.2026.115183

Figure Lengend Snippet: Validation of key PANoptosis-related genes in animal models (A) H&E staining of liver tissues from WT and AILI mice (scale bars, 100 μm; n = 5). (B) mRNA expression of Cdkn1a and Pdk1 by RT-qPCR. (C–F) Correlation analyses between hepatic Cdkn1a and Pdk1 mRNA levels and serum ALT and AST levels at 24 h after AILI. (G) Detection and statistical analysis of key PANoptosis-related gene and marker protein expression in liver tissues from WT and AILI mice. (H) Immunohistochemical staining for P21 and PDK1 in liver tissues from WT and AILI mice (scale bars, 50 μm; n = 5). All the data are presented as the means ± SDs. One-way ANOVA with Tukey’s test and a two-tailed Student’s t test were used for statistical analysis. Spearman’s rank correlation was used to assess the associations between relative mRNA expression levels of Cdkn1a and Pdk1 and serum ALT and AST levels. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: p21 Polyclonal antibody , Proteintech , Cat No.28248-1-AP; RRID: AB_2881097.

Techniques: Biomarker Discovery, Staining, Expressing, Quantitative RT-PCR, Marker, Immunohistochemical staining, Two Tailed Test

Journal: Bone Research

Article Title: Dasatinib and quercetin senolytic treatment delays early onset intervertebral disc degeneration in SM/J mice

doi: 10.1038/s41413-026-00526-4

Figure Lengend Snippet: Caudal discs of SM/J mice evidence early cellular senescence and a senescence signature during degeneration. At four weeks of age, SM/J mice have increase abundance of senescence markers in their caudal discs, evidenced by ( a–a” ) p19 and ( b–b” ) p21 abundance relative to age-matched B6J discs. c Microarray analysis of 4-week-old and 17-week-old SM/J NP and AF shows distinct clustering in both tissues between the two timepoints. Thematic analysis in CompBio of enriched concepts in 17-week-old NP tissues, compared to 4-week-old tissues shows: ( d’ ) Beta-galactoside Alpha-2,3-sialytransferase Activity is an upregulated theme in the NP; ( e ) VEGF-A Complex is an upregulated theme in the AF; ( f ) CDK1 Phosphorylates Condensin is a downregulated theme in the NP; and ( g ) RUNX2 Regulates Osteoblast Differentiation is a downregulated theme in the AF. h Venn Diagram showing the gene-level overlap between SM/J tissue profiles and the SenMayo geneset, with the overlapping genes shown in ( i ). Data are shown as mean ± SD. Significance was determined using an unpaired t -test or Mann-Whitney test, as appropriate. n C57BL/6J 4w = 8; n SM/J 4w = 5; n SM/J 17w = 6

Article Snippet: Tissue sections were then blocked in 2%–10% normal serum in PBS-T, and incubated with antibodies against p19 (1:100, Novus NB200-106), p21 (1:200, Novus NB100-1941), collagen I (1:100, Abcam ab34710), aggrecan (1:50; Millipore; AB1031), chondroitin sulfate (1:300, Abcam ab11570), IL-1b (1:100, Novus NB600-633), IL-6 (1:50, Novus NB600-1131), TGFb (1:100; Abcam; ab92486), collagen X (1:500, Abcam ab58632), CA3 (1:150, Santa Cruz), and GLUT-1 (1:200, Abcam, ab40084).

Techniques: Microarray, Activity Assay, MANN-WHITNEY

Journal: Bone Research

Article Title: Dasatinib and quercetin senolytic treatment delays early onset intervertebral disc degeneration in SM/J mice

doi: 10.1038/s41413-026-00526-4

Figure Lengend Snippet: DQ reduces caudal disc degeneration and senescence in SM/J mice. a , b Schematic showing study design: intraperitoneal injections of DQ, Nav., or a Vehicle control were administered once every week to mice starting at 4 weeks of age and ending at 17 weeks of age. a’ –a’” SafraninO/Fast Green/Hematoxylin staining evaluated with modified Thompson scoring shows DQ improves disc degeneration in SM/J mice. Images reflect the range of degenerative outcomes across treatment cohorts. b’ –b” Safranin/Fast Green/Hematoxylin staining evaluated with modified Thompson scoring shows Nav. does not improve disc degeneration in SM/J mice. Quantitative immunohistochemistry shows reduced ( c–c” ) p19 (NP and AF) and ( d–d” ) p21 (AF only) in DQ-treated SM/J discs. SASP markers of ( e–e” ) TGF β, ( f–f” ) IL-6, ( g–g” ) MMP13, and ( h–h” ) IL-1β indicate DQ mediates SASP in SM/J discs. Data are shown as mean ± SD. Significance was determined using an unpaired t -test or Mann-Whitney test, as appropriate. Distribution statistics were determined using a χ 2 test. 17 weeks old ( n DQ = 11, 6 females + 5 males; n CT = 13, 6 females + 7 males; n Nav. = 7, n Veh. = 7)

Article Snippet: Tissue sections were then blocked in 2%–10% normal serum in PBS-T, and incubated with antibodies against p19 (1:100, Novus NB200-106), p21 (1:200, Novus NB100-1941), collagen I (1:100, Abcam ab34710), aggrecan (1:50; Millipore; AB1031), chondroitin sulfate (1:300, Abcam ab11570), IL-1b (1:100, Novus NB600-633), IL-6 (1:50, Novus NB600-1131), TGFb (1:100; Abcam; ab92486), collagen X (1:500, Abcam ab58632), CA3 (1:150, Santa Cruz), and GLUT-1 (1:200, Abcam, ab40084).

Techniques: Control, Staining, Modification, Immunohistochemistry, MANN-WHITNEY

Journal: Bone Research

Article Title: Dasatinib and quercetin senolytic treatment delays early onset intervertebral disc degeneration in SM/J mice

doi: 10.1038/s41413-026-00526-4

Figure Lengend Snippet: JUN pathway inhibition mimics the positive effect of DQ in decreasing senescence and SASP in human degenerated NP cells. a Grade IV and V human degenerated NP cells show a lower percentage of β-Gal-staining after treatment with DQ and JUN inhibitor, or only DQ, respectively. b Grade IV Human NP cells exhibit lower expression of IL-6 , MMP2 , and MMP13 compared to the stimulus group. Additionally, DQ treatment resulted in a decrease in CDKN1A and IL-6 , while T5224 enhanced MMP13 expression. c Grade V human NP cells exhibit lower expression of CDKN1A, CDKN2A, IL-6 , CCL2 , MMP2 , and MMP13 relative to the stimulus group. DQ treatment attenuated the expression of IL-6 and CCL2 , and MMP2 . T5224 ameliorated CDKN1A , IL-6 , CCL2 , and MMP2 expression. Data are shown as mean ± SD. Significance was determined using Dunnett’s multiple comparisons test ( n = 3 independent experiments, performed in triplicate)

Article Snippet: Tissue sections were then blocked in 2%–10% normal serum in PBS-T, and incubated with antibodies against p19 (1:100, Novus NB200-106), p21 (1:200, Novus NB100-1941), collagen I (1:100, Abcam ab34710), aggrecan (1:50; Millipore; AB1031), chondroitin sulfate (1:300, Abcam ab11570), IL-1b (1:100, Novus NB600-633), IL-6 (1:50, Novus NB600-1131), TGFb (1:100; Abcam; ab92486), collagen X (1:500, Abcam ab58632), CA3 (1:150, Santa Cruz), and GLUT-1 (1:200, Abcam, ab40084).

Techniques: Inhibition, Staining, Expressing