Journal: Bioactive Materials

Article Title: A multimodal ROS logic-gated therapeutic platform disrupts the vicious cycle of senescence to promote aged bone defect repair

doi: 10.1016/j.bioactmat.2026.02.002

Figure Lengend Snippet: Anti-senescence effects of the hydrogel platform on BMSCs. (A) Schematic diagram illustrating hallmark differences between senescent and rejuvenated BMSCs. (B) qRT-PCR analysis of senescence-related genes ( p16 , p21 , p53 ) after different treatments ( n = 3). (C) Western blot analysis of p16, p21, and p53 protein expression in senescent BMSCs. (D) Corresponding quantitative analysis of protein expression ( n = 3). (E) Representative immunofluorescence images of p53 staining in BMSCs (scale bars: 90 μm). (F) Quantitative analysis of p53 fluorescence intensity ( n = 3). (G) Representative cell cycle distribution plots obtained by flow cytometry. (H) Statistical analysis of S-phase ( n = 3). (I) Representative SA- β -gal staining images of senescent BMSCs (scale bars: 200 μm). (J) Quantification of SA- β -gal positive cells ( n = 3). (K) qRT-PCR analysis of SASP-associated genes ( Ccl2 , Mmp2 , Il-6 , Tnf-α ) in BMSCs ( n = 3). Data are presented as mean ± SD; ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Article Snippet: Tissue sections were stained for γ-H2A.X (Servicebio, GB111841 , 1:500), p62 (Affinity, AF5384, 1:500), p16 (Santa Cruz, sc-81156, 1:200), p21 (Affinity, AF6290, 1:300), Cyclin D1 (Proteintech, 26939-1-AP, 1:200), MMP-2 (Servicebio, GB11130, 1:500), IL-6 (Servicebio, GB11117, 1:200), and TNF-α (Servicebio, GB115702 , 1:200) following standard protocols.

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Immunofluorescence, Staining, Fluorescence, 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: 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: Bioactive Materials

Article Title: On-demand mild photothermal cascade platform reprogramming mitochondrial immunity for tendon rejuvenation

doi: 10.1016/j.bioactmat.2026.01.004

Figure Lengend Snippet: LT-NPs-NIR protects TSPCs against oxidative stress-induced senescence and preserves tenogenic phenotype. (A–D) Immunofluorescence staining for DNA damage (γ-H2AX), proliferation (Ki67), and senescence markers (P16, P53). (E–G) Assessment of stemness (SOX2) and tenogenic differentiation markers (SCX, COL1). (H) Quantitative analysis of the indicated markers. (I) qRT-PCR analysis of SASP-related inflammatory mediators (IL-1β, CXCL10) and matrix-degrading enzymes (MMP3, MMP13). (J) Schematic illustrating the mechanism of ROS scavenging and SASP inhibition. Significance: ns, not significant; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Snt: senescent cells; Yng: young cells.

Article Snippet: After washing, cells were incubated with primary antibodies against Ki67 (ab15580, Abcam), Phosphorylated Histone H2AX (γ-H2AX) (ab81299, Abcam), SOX2 (sc-365964, Santa Cruz), Type I Collagen (COL1) (ab138492, Abcam), tenomodulin (TNMD) (ab203676, Abcam; sc-51813, Santa Cruz), Scleraxis (SCX) (sc-518082, Santa Cruz), IRF3 (ab68481, Abcam), Transcription Factor p65/RELA (P65) (A22331, Abclonal), Cyclin-Dependent Kinase Inhibitor 2A (p16INK4a) (P16) (sc-1661, Santa Cruz), P53 (10442-1-AP, Proteintech), Inducible Nitric Oxide Synthase (iNOS) (ab178945, Abcam), Arginase-1(Arg-1) (ab96183, Abcam), HSP70 (sc-32239, Santa Cruz), IL-6 (ab233706, Abcam), Matrix Metalloproteinase 13 (MMP13) (ab39012, Abcam), Double-stranded DNA (dsDNA) Marker (sc-58749, Santa Cruz), and Translocase of Outer Mitochondrial Membrane 20 (TOMM20) (11802-1-AP, Proteintech).

Techniques: Immunofluorescence, Staining, Quantitative RT-PCR, Inhibition

Journal: Bioactive Materials

Article Title: On-demand mild photothermal cascade platform reprogramming mitochondrial immunity for tendon rejuvenation

doi: 10.1016/j.bioactmat.2026.01.004

Figure Lengend Snippet: LT-NPs-NIR modulate macrophage polarization and enhance TSPC-mediated tenogenic repair in a Transwell co-culture system. (A) Schematic of the Transwell co-culture setup. (B) SA-β-gal staining of macrophages. (C–F) Immunofluorescence of TSPCs for (C) P16, (D) SOX2, (E) SCX, and (F) Tenomodulin (TNMD) with F-actin. (G) Quantification of P16, SOX2, SCX, and TNMD levels. (H) Proposed mechanism: LT-NPs-NIR promote an M1-to-M2 macrophage shift and regulate TSPC senescence/stemness balance to favor tenogenic repair, potentially via STING/NF-κB signaling. Scale bars: 100 μm (B); 50 μm (C–E); 100 μm (F). Significance: ns, not significant; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: After washing, cells were incubated with primary antibodies against Ki67 (ab15580, Abcam), Phosphorylated Histone H2AX (γ-H2AX) (ab81299, Abcam), SOX2 (sc-365964, Santa Cruz), Type I Collagen (COL1) (ab138492, Abcam), tenomodulin (TNMD) (ab203676, Abcam; sc-51813, Santa Cruz), Scleraxis (SCX) (sc-518082, Santa Cruz), IRF3 (ab68481, Abcam), Transcription Factor p65/RELA (P65) (A22331, Abclonal), Cyclin-Dependent Kinase Inhibitor 2A (p16INK4a) (P16) (sc-1661, Santa Cruz), P53 (10442-1-AP, Proteintech), Inducible Nitric Oxide Synthase (iNOS) (ab178945, Abcam), Arginase-1(Arg-1) (ab96183, Abcam), HSP70 (sc-32239, Santa Cruz), IL-6 (ab233706, Abcam), Matrix Metalloproteinase 13 (MMP13) (ab39012, Abcam), Double-stranded DNA (dsDNA) Marker (sc-58749, Santa Cruz), and Translocase of Outer Mitochondrial Membrane 20 (TOMM20) (11802-1-AP, Proteintech).

Techniques: Co-Culture Assay, Staining, Immunofluorescence

Journal: Bioactive Materials

Article Title: On-demand mild photothermal cascade platform reprogramming mitochondrial immunity for tendon rejuvenation

doi: 10.1016/j.bioactmat.2026.01.004

Figure Lengend Snippet: Molecular assessment of tendon repair and systemic biosafety. (A) Representative immunofluorescence images of inflammatory, matrix-degrading, tenogenic, and senescence markers, alongside macrophage phenotypes in repaired tendons. (B) Correlation analysis integrating molecular and functional recovery. Bar charts (left Y-axis) display the relative fluorescence intensity of the indicated markers, overlaid with line graphs (right Y-axis) showing the fold change in biomechanical properties (Ultimate Load and Tensile Modulus). Note the inverse correlation between SASP factors (IL-6, MMP13, P16) and mechanical strength. (C) Western blot analysis of the STING pathway, senescence indicators, and heterotopic ossification markers (OCN, SOX9, BMP-2). (D, E) Systemic biosafety evaluation via H&E staining of major organs (D) and blood biochemistry analysis (E) showing no toxicity. Significance: ns, not significant; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: After washing, cells were incubated with primary antibodies against Ki67 (ab15580, Abcam), Phosphorylated Histone H2AX (γ-H2AX) (ab81299, Abcam), SOX2 (sc-365964, Santa Cruz), Type I Collagen (COL1) (ab138492, Abcam), tenomodulin (TNMD) (ab203676, Abcam; sc-51813, Santa Cruz), Scleraxis (SCX) (sc-518082, Santa Cruz), IRF3 (ab68481, Abcam), Transcription Factor p65/RELA (P65) (A22331, Abclonal), Cyclin-Dependent Kinase Inhibitor 2A (p16INK4a) (P16) (sc-1661, Santa Cruz), P53 (10442-1-AP, Proteintech), Inducible Nitric Oxide Synthase (iNOS) (ab178945, Abcam), Arginase-1(Arg-1) (ab96183, Abcam), HSP70 (sc-32239, Santa Cruz), IL-6 (ab233706, Abcam), Matrix Metalloproteinase 13 (MMP13) (ab39012, Abcam), Double-stranded DNA (dsDNA) Marker (sc-58749, Santa Cruz), and Translocase of Outer Mitochondrial Membrane 20 (TOMM20) (11802-1-AP, Proteintech).

Techniques: Immunofluorescence, Functional Assay, Fluorescence, Western Blot, Staining

Journal: Biogerontology

Article Title: Ageing-related structural and cellular alterations in the mouse muscle-tendon junction

doi: 10.1007/s10522-026-10428-x

Figure Lengend Snippet: Senescence was detected more frequently in MTJ-specific and vascular endothelial cells compared to the overall cell population. Representative in situ hybridisation images of young and old mouse MTJ (n = 4 per age group) showing senescence markers ( a ) p16 and ( b ) p21.Red: Col22 (MTJ marker); yellow: ( a ) p16 and ( b ) p21; cyan: VWF (endothelial cell marker); grey: DAPI. Blue DAPI image on the right shows the interface between the muscle and tendon indicated by orange dashed lines. Scale bar is 50 µm. Percentage of ( c ) p16 and ( d ) p21 positive cells were measured in Col22-positive MTJ-specific cells, VWF-positive endothelial cells, and all cells in the field of view. Y: young; O: old. Two-way ANOVA followed by Tukey’s multiple comparisons test was used to calculate the significance between the age groups and cell populations. Data are presented as mean ± SD: *p < 0.05 **p < 0.01 ****p < 0.0001

Article Snippet: The details of primary antibodies were as follows: Guinea‐pig anti‐collagen XXII (1:100, monoclonal, a kind gift from Manuel Koch, University of Cologne, Germany) and rabbit anti-p16 (1:50, 80772S, Cell Signaling Technology, MA, USA).

Techniques: In Situ, Hybridization, Marker

Journal: Biogerontology

Article Title: Ageing-related structural and cellular alterations in the mouse muscle-tendon junction

doi: 10.1007/s10522-026-10428-x

Figure Lengend Snippet: Protein expression analysis showed an increase in p16-positive cell proportion within Col22-positive MTJ-specific cells with age. a Representative immunofluorescent images of young and old MTJ (n = 4 per age group). Red: Col22; green: p16; grey: DAPI. Blue DAPI image on the right shows the interface between the muscle and tendon indicated by orange dashed lines. Scale bar is 50 µm. b Percentage of p16 positive cells in Col22-positive MTJ-specific cell population. Data are presented as mean ± SD (n = 4). A Mann-Whitney test was used to calculate the significance between young and old tendons. *p < 0.05

Article Snippet: The details of primary antibodies were as follows: Guinea‐pig anti‐collagen XXII (1:100, monoclonal, a kind gift from Manuel Koch, University of Cologne, Germany) and rabbit anti-p16 (1:50, 80772S, Cell Signaling Technology, MA, USA).

Techniques: Expressing, MANN-WHITNEY

Journal: International Journal of Oncology

Article Title: Overcoming acquired doxorubicin resistance of ovarian carcinoma cells by verapamil-mediated promotion of DNA damage-driven cytotoxicity

doi: 10.3892/ijo.2026.5861

Figure Lengend Snippet: Influence of combined treatment of A2780ADR cells with Doxo and selected inhibitors on mechanism of the DDR and mRNA expression of selected susceptibility-related genes. (A) Logarithmically growing A2780ADR cells were co-treated with the indicated concentrations of Doxo and selected pharmacological inhibitors (concentrations see ) for 24 or 72 h. Afterwards, the protein expression of DDR-related factors was analyzed by western blotting. For loading control, blots were reprobed with ERK2 antibody. (B) Reverse transcription-quantitative PCR of the mRNA expression of selected factors known to contribute to different mechanisms of drug sensitivity. Data shown are mean ± SD from triplicate determinations as described in methods. Relative mRNA level in untreated A2780ADR cells was set to 1.0. Doxo, doxorubicin; DDR, DNA damage response; p-, phosphorylated; nd, not detectable; Bax, Bcl-2 associated protein X; Bcl-2, B-cell lymphoma; BBC3, Bcl-2 binding component 2; BRCA1, 2, breast cancer associated gene 1,2; Cl casp-7, cleaved caspase 7; Chk, checkpoint kinase; CXCL8, chemokine ligand 8 (interleukin 8); p21, CDK inhibitor 1; p16, CDK inhibitor 2; CDKN1A/2A, cyclin dependent kinae inhibitor 1A/2A; CCNB1, Cyclin B1; b-Gal, beta-galactosidase; FASL, FAS ligand; FASR, FAS receptor; GADD, growth arrest and DNA damage inducible GPX1, glutathione peroxidase 1; GSTM1, glutathione S-transferase 1; HMOX1, heme oxygenase 1; γH2AX, Ser139 phosphorylated histone H2AX; p53, tumor suppressor p53; PARP, poly (ADP-ribose) polymerase; PCNA-proliferating cell nuclear antigen; PGC1A, PPARG coactivator 1; PPARGC1A, peroxisome proliferator-activated receptor gamma coactivator 1-alpha; RAD51, radiation damage gene 51; RPAreplication protein A; SOD1, superoxide dismutase 1; Ver, verapamil.

Article Snippet: The following primary antibodies were used: Copper transporting ATPase (ATP7A), extracellular regulated kinase 2 (ERK2), phosphorylated (p)-histone H3 (Ser10) from Thermo Fisher Scientific Inc., cleaved caspase-7 (Asp198), p-Chk1 (Ser 345), cyclin B1, galactosidase β (E2U2I), GAPDH (14C10), MDR1/ABCB1 (D3H1Q), p-P53 (S15), PARP, TopBP1(D8G4L), topoisomerase IIa (D10G9), 53BP1 and Ki67 were from Cell Signaling Technology Inc., pChk2 (T68) [Y171], copper uptake protein 1 (CTR1/SLC31A1) [EPR7936] and Rad51 from Abcam, γH2AX (Ser 139) clone JBW301, p-KAP-1 (S824) and p-RPA32 (S4/S8) from Bethyl Laboratories Inc., organic cation transporter-2 (OCT2) from Biozol Diagnostics Vertrieb GmbH, p16 (F-12) and p21 (C-19) from Santa Cruz Biotechnology, Inc. As secondary antibodies, horseradish peroxidase-conjugated secondary antibodies goat anti-mouse IgG and mouse anti-rabbit IgG were used (Rockland Immunochemicals Inc.).

Techniques: Expressing, Western Blot, Control, Reverse Transcription, Real-time Polymerase Chain Reaction, Binding Assay

Journal: Aging and Disease

Article Title: The Flip Side of the Coin: METTL3 Serves as a Novel Cellular Senescence Accelerator via Negative Regulation of ITGA9

doi: 10.14336/AD.2024.1715

Figure Lengend Snippet: M6A modifications within total mRNA were significantly enhanced during the aging process . ( A ) Left: representative photographs of 2-month and 20-month-old male C57BL/6 mouse; Right: representative photographs of SA-β-gal staining in kidney tissues from young and old C57-BL/6 mice. ( B ) Left: representative photographs of SA-β-gal staining of P3 and P9 MEF cells; Right: the p16 expression levels in P3 and P9 MEF cells. ( C ) Left: representative photographs of SA-β-gal staining of H 2 O 2 -induced premature senescence of NIH/3T3 cells; Right: the p16 expression levels in H 2 O 2 treated NIH/3T3 cells. ( D ) Poly(A)+ RNA was extracted from the brain and liver tissue of young and old C57-BL/6 mice and subjected to RNA dot-blot analysis with an antibody recognizing m6A. Methylene blue staining served as the loading control. ( E ) Poly(A)+ RNA was extracted from the P3 and P9 MEF cells and subjected to RNA dot-blot analysis with an antibody recognizing m6A. Methylene blue staining served as the loading control. ( F ) Poly(A)+ RNA was extracted from the P3 and P9 MEF cells and subjected to RNA dot-blot analysis with an antibody recognizing m6A. Methylene blue staining served as the loading control. ( G ) Expression levels of m6A modification associated proteins were measured in P3 and P9 MEF cells, and the quantitative results were shown on the right. Mean (±SEM), n=4, passed normality test: Shapiro-Wilk test; One sample t test (MEF P9 vs 1, METTL3: p =0.0027, t = 9.176).

Article Snippet: M6A antibody (202003, Synaptic Systems, Germany), GAPDH Monoclonal antibody (60004-1-Ig, Proteintech, China), METTL3 Monoclonal antibody (ab195352, Abcam, UK), FTO Polyclonal antibody (27226-1-AP, Proteintech, China), METTL14 Polyclonal antibody (AP22363a, Abgent, USA), ALKBH5 Polyclonal antibody (16837-1-AP, Proteintech, China), WTAP Monoclonal antibody (60188-1-Ig, Proteintech, China), p16 INK4A antibody (F-12) (sc-1661, Santa Cruz, USA), ITGA9 Polyclonal antibody (AF3827, R&D, USA), BrdU Monoclonal antibody (66241-1-Ig, Proteintech, China), Goat Anti-Mouse antibody (Alexa Fluor® 488) (SA00013-1, Proteintech, China), Goat anti-rabbit IgG (H+L) antibody (A0208, Beyotime, China), Goat anti-mouse IgG (H+L) antibody (A0216, Beyotime, China), Donkey anti-goat IgG (H+L) antibody (A0181, Beyotime, China).

Techniques: Staining, Expressing, Dot Blot, Control, Modification

Journal: Aging and Disease

Article Title: The Flip Side of the Coin: METTL3 Serves as a Novel Cellular Senescence Accelerator via Negative Regulation of ITGA9

doi: 10.14336/AD.2024.1715

Figure Lengend Snippet: METTL3 accelerated replicative senescence in MEFs . ( A ) The protein levels in MEFs transfected with siNC and siMETTL3-1/2/3. Mean (±SEM), n=3, passed normality test: Shapiro-Wilk test; One sample t test (siMETTL3-1 vs 1: p =0.0113, t =9.328; siMETTL3-2VS 1: p =0.0022, t =21.05; siMETTL3-3 vs 1: p =0.0117, t = 9.155). ( B ) Poly(A)+ RNA was extracted from MEF cells transfected with siNC and siMETTL3 and subjected to RNA dot-blot analysis with an antibody recognizing m6A. Methylene blue staining served as the loading control. ( C ) Western blot assay of p16 expression in MEFs transfected with siNC and siMETTL3. The quantitative results were shown on the right. Mean (±SEM), n=4, passed normality test: Shapiro-Wilk test; One sample t test (siMETTL3 vs 1, p =0.0006, t =15.36). ( D ) Representative photographs of SA-β-gal staining of MEF cells transfected with siNC and siMETTL3 (×100). The quantitative results of SA-β-gal staining were shown on the right. Mean (±SEM), n=4, passed normality test: Shapiro-Wilk test; Paired t test (siMETTL3 vs siNC, p =0.0025, t =9.407). ( E ) Representative photographs of cells stained with DAPI (blue fluorescence) and BrdU (green fluorescence) in siNC and siMETTL3 transfected MEF cells (scale bar: 400μm). The quantitative results of the ratio (BrdU stained cells / DAPI stained cells) were shown on the right. Mean (±SEM), n=4, passed normality test: Shapiro-Wilk test; Paired t test (siMETTL3 vs siNC, p =0.009, t =6.065). ( F ) The mRNA levels in MEFs infected with Lenti-NC and METTL3 lentivirus. Mean (±SEM), n=3, passed normality test: Shapiro-Wilk test; One sample t test (METTL3VS 1, p =0.0058, t =13.03). ( G ) Poly(A)+ RNA was extracted from MEF cells infected with Lenti-NC and METTL3 lentivirus and subjected to RNA dot-blot analysis with an antibody recognizing m6A. Methylene blue staining served as the loading control. ( H ) Western blot assay of METTL3 and p16 expression in MEFs infected with METTL3 lentivirus. The quantitative results were shown on the right. Mean (±SEM), n=3, passed normality test: Shapiro-Wilk test; One sample t test (METTL3 vs 1, p =0.0342, t =5.271). ( I ) Representative photographs of SA-β-gal staining of MEF cells infected with Lenti-NC and METTL3 lentivirus (×100). The quantitative results of SA-β-gal staining were shown on the right. Mean (±SEM), n=3, passed normality test: Shapiro-Wilk test; Paired t test (METTL3 vs Lenti-NC, p =0.0088, t =10.59). ( J ) Representative photographs of cells stained with DAPI (blue fluorescence) and BrdU (green fluorescence) in Lenti-NC and METTL3 lentivirus infected MEF cells (scale bar: 400μm). The quantitative results of the ratio (BrdU stained cells / DAPI stained cells) were shown on the right. Mean (±SEM), n=3, passed normality test: Shapiro-Wilk test; Paired t test (METTL3 vs Lenti-NC, p =0.0065, t =12.35).

Article Snippet: M6A antibody (202003, Synaptic Systems, Germany), GAPDH Monoclonal antibody (60004-1-Ig, Proteintech, China), METTL3 Monoclonal antibody (ab195352, Abcam, UK), FTO Polyclonal antibody (27226-1-AP, Proteintech, China), METTL14 Polyclonal antibody (AP22363a, Abgent, USA), ALKBH5 Polyclonal antibody (16837-1-AP, Proteintech, China), WTAP Monoclonal antibody (60188-1-Ig, Proteintech, China), p16 INK4A antibody (F-12) (sc-1661, Santa Cruz, USA), ITGA9 Polyclonal antibody (AF3827, R&D, USA), BrdU Monoclonal antibody (66241-1-Ig, Proteintech, China), Goat Anti-Mouse antibody (Alexa Fluor® 488) (SA00013-1, Proteintech, China), Goat anti-rabbit IgG (H+L) antibody (A0208, Beyotime, China), Goat anti-mouse IgG (H+L) antibody (A0216, Beyotime, China), Donkey anti-goat IgG (H+L) antibody (A0181, Beyotime, China).

Techniques: Transfection, Dot Blot, Staining, Control, Western Blot, Expressing, Fluorescence, Infection

Journal: Aging and Disease

Article Title: The Flip Side of the Coin: METTL3 Serves as a Novel Cellular Senescence Accelerator via Negative Regulation of ITGA9

doi: 10.14336/AD.2024.1715

Figure Lengend Snippet: ITGA9 staved off senescence in MEFs . ( A ) The mRNA and protein levels of ITGA9 in MEF cells transfected with siNC and siITGA9-1/2/3.Mean (±SEM), n=3, passed normality test: Shapiro-Wilk test; One sample t test (siITGA9-1 vs 11: p =0.0002, t =68.12; siITGA9-2 vs 1: p =0.0003, t =63.15 ; siITGA9-3 vs 11: p =0.0111, t =9.410; ) (B) The p16 expression levels in MEF cells transfected with siNC and siITGA9. The quantitative results were shown on the right. Mean (±SEM), n=3, passed normality test: Shapiro-Wilk test; One sample t test (siITGA9 vs 1, p =0.0012, t =28.41). ( C ) Representative photographs of SA-β-gal staining of MEF cells transfected with siNC and siITGA9 (×100). The quantitative results of SA-β-gal staining were shown on the right. Mean (±SEM), n=3, passed normality test: Shapiro-Wilk test; Paired t test (siITGA9 vs siNC, p =0.0026, t =19.65). ( D ) Representative photographs of cells stained with DAPI (blue fluorescence) and BrdU (green fluorescence) in siNC and siITGA9 transfected MEF cells (scale bar: 400μm). The quantitative results of the ratio ( BrdU stained cells / DAPI stained cells) were shown on the right. Mean (±SEM), n=3, passed normality test: Shapiro-Wilk test; Paired t test (siITGA9 vs siNC, p =0.0144, t =4.138). ( E ) Representative photographs of cells stained with DAPI (blue fluorescence) and BrdU (green fluorescence) in Lenti-NC and ITGA9 infected MEF cells (scale bar: 400μm). The quantitative results of the ratio ( BrdU stained cells / DAPI stained cells) were shown on the right. Mean (±SEM), n=4, passed normality test: Shapiro-Wilk test; Paired t test (ITGA9 vs Lenti-NC, p =0.01, t =5.843). ( F ) The mRNA levels of ITGA9 in MEF cells infected with Lenti-NC or ITGA9 lentivirus. Mean (±SEM), n=3, passed normality test: Shapiro-Wilk test; One sample t test (ITGA9 vs 1, p =0.0025, t =20.08). ( G ) The ITGA9 and p16 protein expression levels in MEFs infected with Lenti-NC and ITGA9 lentivirus. The results of grayscale scanning were shown on the right. Mean (±SEM), n=3, passed normality test: Shapiro-Wilk test; One sample t test (ITGA9 vs 1, p =0.0256, t =6.124). ( H ) Representative photographs of SA-β-gal staining of MEF cells infected with Lenti-NC and ITGA9 lentivirus (×100). The quantitative results of SA-β-gal staining were shown on the right. Mean (±SEM), n=3, passed normality test: Shapiro-Wilk test; Paired t test (ITGA9 vs Lenti-NC, p =0.0009, t =33.30).

Article Snippet: M6A antibody (202003, Synaptic Systems, Germany), GAPDH Monoclonal antibody (60004-1-Ig, Proteintech, China), METTL3 Monoclonal antibody (ab195352, Abcam, UK), FTO Polyclonal antibody (27226-1-AP, Proteintech, China), METTL14 Polyclonal antibody (AP22363a, Abgent, USA), ALKBH5 Polyclonal antibody (16837-1-AP, Proteintech, China), WTAP Monoclonal antibody (60188-1-Ig, Proteintech, China), p16 INK4A antibody (F-12) (sc-1661, Santa Cruz, USA), ITGA9 Polyclonal antibody (AF3827, R&D, USA), BrdU Monoclonal antibody (66241-1-Ig, Proteintech, China), Goat Anti-Mouse antibody (Alexa Fluor® 488) (SA00013-1, Proteintech, China), Goat anti-rabbit IgG (H+L) antibody (A0208, Beyotime, China), Goat anti-mouse IgG (H+L) antibody (A0216, Beyotime, China), Donkey anti-goat IgG (H+L) antibody (A0181, Beyotime, China).

Techniques: Transfection, Expressing, Staining, Fluorescence, Infection

Journal: Aging and Disease

Article Title: The Flip Side of the Coin: METTL3 Serves as a Novel Cellular Senescence Accelerator via Negative Regulation of ITGA9

doi: 10.14336/AD.2024.1715

Figure Lengend Snippet: METTL3 accelerated cellular senescence by modulating ITGA9 . ( A ) The p16 level in MEF cells transfected with siNC, siMETTL3 or siMETTL3 and siITGA9. The quantitative results were shown on the right. Mean (±SEM), n=4, passed normality test: Shapiro-Wilk test; Paired t test ( p =0.0058, t =7.062). ( B ) The p16 level in MEF cells infected with Lenti-NC, METTL3 or METTL3 and ITGA9. The quantitative results were shown on the right. Mean (±SEM), n=3, passed normality test: Shapiro-Wilk test; Paired t test ( p =0.017, t =7.563). (C, D) Representative photographs of SA-β-gal staining (C) and BrdU incorporation staining (D) of MEF cells transfected with siNC, siMETTL3, siITGA9, or siMETTL3 and siITGA9. The quantitative results were shown on the right. Mean (±SEM), passed normality test: Shapiro-Wilk test; Paired t test (C), n=3, p =0.0044, t =15.02; (D), n=4, p =0.0003, t =19.14). (E, F) Representative photographs of SA-β-gal staining (E) and BrdU incorporation staining (F) of MEF cells infected with Lenti-NC, METTL3, ITGA9 or METTL3 and ITGA9. The quantitative results were shown on the right. Mean (±SEM), n=3, passed normality test: Shapiro-Wilk test; Paired t test ((E): p =0.0095, t =10.18; (F): p =0.0231, t =6.469).

Article Snippet: M6A antibody (202003, Synaptic Systems, Germany), GAPDH Monoclonal antibody (60004-1-Ig, Proteintech, China), METTL3 Monoclonal antibody (ab195352, Abcam, UK), FTO Polyclonal antibody (27226-1-AP, Proteintech, China), METTL14 Polyclonal antibody (AP22363a, Abgent, USA), ALKBH5 Polyclonal antibody (16837-1-AP, Proteintech, China), WTAP Monoclonal antibody (60188-1-Ig, Proteintech, China), p16 INK4A antibody (F-12) (sc-1661, Santa Cruz, USA), ITGA9 Polyclonal antibody (AF3827, R&D, USA), BrdU Monoclonal antibody (66241-1-Ig, Proteintech, China), Goat Anti-Mouse antibody (Alexa Fluor® 488) (SA00013-1, Proteintech, China), Goat anti-rabbit IgG (H+L) antibody (A0208, Beyotime, China), Goat anti-mouse IgG (H+L) antibody (A0216, Beyotime, China), Donkey anti-goat IgG (H+L) antibody (A0181, Beyotime, China).

Techniques: Transfection, Infection, Staining, BrdU Incorporation Assay