p21  (Bioss)

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: Journal of Translational Medicine

Article Title: LDHC4 drives lung adenocarcinoma progression by inducing lactylation of RB1 at lysine 900 to disrupt the RB1–E2F1 complex

doi: 10.1186/s12967-026-08070-9

Figure Lengend Snippet: Influence of RB1–K900lac on the cell cycle pathway. ( A to C ) Cell cycle analysis by flow cytometry in A549 and PC-9 cell lines stably expressing RB1–WT or RB1–K900R via lentiviral vectors. ( D ) Representative immunofluorescence images showing the distribution of CDK1 in A549 and PC-9 cells. CDK1 protein was labeled with red fluorescent Cy3, and nuclei were counterstained with blue fluorescent DAPI. Images were acquired using a high-resolution confocal multiphoton microscopy system (NIKON AX RMP, Japan). ( E , F ) Western blot analysis of cell cycle-related CDK molecule expression. ( G , H ) Expression of cell cycle-related cyclin molecules. ( I , J ) Expression of P21 and Chk1 molecules. ( K ) Schematic diagram illustrating how LDHC4 promotes the cell cycle by inducing RB1 lactylation. ** p < 0.01, *** p < 0.001

Article Snippet: The following antibodies were used in this study: rabbit anti-human LDHC (subunit C) monoclonal antibody (mAb) (Proteintech Group, Inc., 1:1000), rabbit anti-human RB1 mAb (Proteintech Group, Inc., 1:2000), rabbit anti-human L-Lactyl Lysine mAb (PTM Bio, Inc., 1:1000), rabbit anti-human E2F1 mAb (APExBIO Technology, LLC, 1:500), rabbit anti-human Lamin B mAb (Beyotime Biotech, Inc., 1:1000), rabbit anti-human CDK1 mAb (Beyotime Biotech, Inc., 1:800), rabbit anti-human CDK2 mAb (Beyotime Biotech, Inc., 1:800), rabbit anti-human CDK4 mAb (Beyotime Biotech, Inc., 1:1000), rabbit anti-human CDK6 mAb (Beyotime Biotech, Inc., 1:1000), rabbit anti-human cyclin A2 mAb (Beyotime Biotech, Inc., 1:1000), rabbit anti-human cyclin B1 mAb (Beyotime Biotech, Inc., 1:1000), rabbit anti-human cyclin D1 mAb (Beyotime Biotech, Inc., 1:500), rabbit anti-human P21 mAb (Abmart Bio, Inc., 1:500), rabbit anti-human Chk1 mAb (Immunoway Bio, Inc., 1:2000), and rabbit anti-β-Actin monoclonal antibody (Beyotime Biotech, Inc., 1:1000). β-Actin and Lamin B served as loading controls, and protein band intensities were quantified using Image J software.

Techniques: Cell Cycle Assay, Flow Cytometry, Stable Transfection, Expressing, Immunofluorescence, Labeling, Microscopy, Western Blot

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