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rabbit anti p16  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit anti p16
    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 ) <t>p16</t> 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
    Rabbit Anti P16, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 187 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/p16+ink4a/p16+INK4A+Rabbit+mAb/pmc13050345-89-24-28
    Average 96 stars, based on 187 article reviews
    rabbit anti p16 - by Bioz Stars, 2026-09
    96/100 stars

    Images

    1) Product Images from "Ageing-related structural and cellular alterations in the mouse muscle-tendon junction"

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

    Journal: Biogerontology

    doi: 10.1007/s10522-026-10428-x

    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
    Figure Legend 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

    Techniques Used: In Situ, Hybridization, Marker

    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
    Figure Legend 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

    Techniques Used: Expressing, MANN-WHITNEY

    Related Articles

    Control:

    Article Title: Disordered DNA methylation leads to targetable transcriptional plasticity in ATRT.
    Article Snippet: .. Protein levels were assessed using the following antibodies: IFITM3 (1:1000; Cell Signaling Technology; #59212S), IRF-7 (1:200; Cell Signaling Technology #13014), GAPDH control (1:10,000; Santa Cruz; #SC-47724), STING (1:200; Cell Signaling Technology, #13647S), alphatubulin (1:1,000; #2144S) and p16 INK4A (1:100, Cell Signaling Technology #80772S). ..

    Article Title: Disordered DNA methylation leads to targetable transcriptional plasticity in ATRT
    Article Snippet: .. Protein levels were assessed using the following antibodies: IFITM3 (1:1000; Cell Signaling Technology; #59212S), IRF-7 (1:200; Cell Signaling Technology #13014), GAPDH control (1:10,000; Santa Cruz; #SC-47724), STING (1:200; Cell Signaling Technology, #13647S), alpha-tubulin (1:1,000; #2144S) and p16 INK4A (1:100, Cell Signaling Technology #80772S). ..

    Membrane:

    Article Title: TRIM37–PARP1–TET1 axis maintains stemness and prevents osteoporosis by inhibiting DNMT1 alternative splicing via 5hmC regulation
    Article Snippet: Cell extracts were prepared with M-PER (Pierce, Rockford, IL) or Subcellular Protein Fractionation Kit (Thermo Scientific, Waltham, MA) plus protease inhibitor cocktail (HaltTM; Pierce, Rockford, IL) and protein concentrations were determined using the BCA assay (Pierce, Rockford, IL). .. Aliquots of protein lysates were separated on SDS–8 or 15% polyacrylamide gels and transferred to PVDF membrane filters, which were blocked with 5% blotting grade milk (Bio-Rad, Hercules, CA) in TBST (20 mM Tris-HCl [pH 7.6], 137 mM NaCl, 0.1% Tween 20) for 1 h. The filters were then incubated 1 h at room temperature with a 1:1000 dilution in TBST of antibodies against TET1 (GTX627420, GeneTex), TET2 (ab94580, Abcam, Cambridge, UK), PARP1 (#9532, Cell Signaling Technology), DNMT1 (#5032, Cell Signaling Technology, Danvers, MA), pSer1981-ATM (GTX132146, Gene Tex), TRIM37 (GTX114565, GeneTex), p16 Ink4a (#92803, Cell Signaling Technology, Danvers, MA), p21 Waf1/Cip1 (sc-6246, Santa Cruz Biotechnology), CTCF (GTX65926), HA tag (GTX115044), His tag (GTX628914), and Flag tag (GTX115043, GeneTex), reacted with corresponding secondary antibodies, and detected using a chemiluminescence assay (Millipore, Billerica, MA). ..

    Incubation:

    Article Title: TRIM37–PARP1–TET1 axis maintains stemness and prevents osteoporosis by inhibiting DNMT1 alternative splicing via 5hmC regulation
    Article Snippet: Cell extracts were prepared with M-PER (Pierce, Rockford, IL) or Subcellular Protein Fractionation Kit (Thermo Scientific, Waltham, MA) plus protease inhibitor cocktail (HaltTM; Pierce, Rockford, IL) and protein concentrations were determined using the BCA assay (Pierce, Rockford, IL). .. Aliquots of protein lysates were separated on SDS–8 or 15% polyacrylamide gels and transferred to PVDF membrane filters, which were blocked with 5% blotting grade milk (Bio-Rad, Hercules, CA) in TBST (20 mM Tris-HCl [pH 7.6], 137 mM NaCl, 0.1% Tween 20) for 1 h. The filters were then incubated 1 h at room temperature with a 1:1000 dilution in TBST of antibodies against TET1 (GTX627420, GeneTex), TET2 (ab94580, Abcam, Cambridge, UK), PARP1 (#9532, Cell Signaling Technology), DNMT1 (#5032, Cell Signaling Technology, Danvers, MA), pSer1981-ATM (GTX132146, Gene Tex), TRIM37 (GTX114565, GeneTex), p16 Ink4a (#92803, Cell Signaling Technology, Danvers, MA), p21 Waf1/Cip1 (sc-6246, Santa Cruz Biotechnology), CTCF (GTX65926), HA tag (GTX115044), His tag (GTX628914), and Flag tag (GTX115043, GeneTex), reacted with corresponding secondary antibodies, and detected using a chemiluminescence assay (Millipore, Billerica, MA). ..

    Article Title: The glycolytic enzyme PFKFB3 alleviates DNA damage and chondrocyte senescence in osteoarthritis
    Article Snippet: Secondary antibody, goat anti-rabbit (Yeasen, 33101ES60; dilution 1:200), was incubated for 1 h. Images were taken using a Digital Pathology Scanner (Leica, Wetzlar, Germany). .. In immunofluorescence, after antigen retrieval, primary antibodies against γH2AX (Abcam, ab181861; dilution 1:100), PFKFB3 (Cell Signaling Technology, D7H4Q; dilution 1:50), p16 INK4a (Cell Signaling Technology, #29271; 1:100), p21 (Santa Cruz, sc-397; 1:50), p-ATM (Cell Signaling Technology, #4526; dilution 1:100), p-ATR (Cell Signaling Technology, #30632; dilution 1:100) were incubated overnight. .. Then, slides were incubated with Alexa Fluor 488-conjugated secondary antibodies (Jackson ImmunoResearch, 111-545-003; dilution 1:500) for 1 h and 4′,6-diamidino-2-phenylindole (DAPI; Beyotime) for another 10 min at room temperature.

    FLAG-tag:

    Article Title: TRIM37–PARP1–TET1 axis maintains stemness and prevents osteoporosis by inhibiting DNMT1 alternative splicing via 5hmC regulation
    Article Snippet: Cell extracts were prepared with M-PER (Pierce, Rockford, IL) or Subcellular Protein Fractionation Kit (Thermo Scientific, Waltham, MA) plus protease inhibitor cocktail (HaltTM; Pierce, Rockford, IL) and protein concentrations were determined using the BCA assay (Pierce, Rockford, IL). .. Aliquots of protein lysates were separated on SDS–8 or 15% polyacrylamide gels and transferred to PVDF membrane filters, which were blocked with 5% blotting grade milk (Bio-Rad, Hercules, CA) in TBST (20 mM Tris-HCl [pH 7.6], 137 mM NaCl, 0.1% Tween 20) for 1 h. The filters were then incubated 1 h at room temperature with a 1:1000 dilution in TBST of antibodies against TET1 (GTX627420, GeneTex), TET2 (ab94580, Abcam, Cambridge, UK), PARP1 (#9532, Cell Signaling Technology), DNMT1 (#5032, Cell Signaling Technology, Danvers, MA), pSer1981-ATM (GTX132146, Gene Tex), TRIM37 (GTX114565, GeneTex), p16 Ink4a (#92803, Cell Signaling Technology, Danvers, MA), p21 Waf1/Cip1 (sc-6246, Santa Cruz Biotechnology), CTCF (GTX65926), HA tag (GTX115044), His tag (GTX628914), and Flag tag (GTX115043, GeneTex), reacted with corresponding secondary antibodies, and detected using a chemiluminescence assay (Millipore, Billerica, MA). ..

    Chemiluminescence Immunoassay:

    Article Title: TRIM37–PARP1–TET1 axis maintains stemness and prevents osteoporosis by inhibiting DNMT1 alternative splicing via 5hmC regulation
    Article Snippet: Cell extracts were prepared with M-PER (Pierce, Rockford, IL) or Subcellular Protein Fractionation Kit (Thermo Scientific, Waltham, MA) plus protease inhibitor cocktail (HaltTM; Pierce, Rockford, IL) and protein concentrations were determined using the BCA assay (Pierce, Rockford, IL). .. Aliquots of protein lysates were separated on SDS–8 or 15% polyacrylamide gels and transferred to PVDF membrane filters, which were blocked with 5% blotting grade milk (Bio-Rad, Hercules, CA) in TBST (20 mM Tris-HCl [pH 7.6], 137 mM NaCl, 0.1% Tween 20) for 1 h. The filters were then incubated 1 h at room temperature with a 1:1000 dilution in TBST of antibodies against TET1 (GTX627420, GeneTex), TET2 (ab94580, Abcam, Cambridge, UK), PARP1 (#9532, Cell Signaling Technology), DNMT1 (#5032, Cell Signaling Technology, Danvers, MA), pSer1981-ATM (GTX132146, Gene Tex), TRIM37 (GTX114565, GeneTex), p16 Ink4a (#92803, Cell Signaling Technology, Danvers, MA), p21 Waf1/Cip1 (sc-6246, Santa Cruz Biotechnology), CTCF (GTX65926), HA tag (GTX115044), His tag (GTX628914), and Flag tag (GTX115043, GeneTex), reacted with corresponding secondary antibodies, and detected using a chemiluminescence assay (Millipore, Billerica, MA). ..

    Labeling:

    Article Title: Noninvasive Cell Population Profiling of Normal and Dysplastic Cervical Biofluids by Multicolor Flow Cytometry as a Promising Tool for Companion Diagnostics
    Article Snippet: .. Following directly labeled antibodies were used: p16 INK4a (Cell Signaling, 43161S), Ki-67 (Biolegend, 350526), EpCAM (Biolegend, 324218), CD45 (Biolegend, 304042) and Cytokeratin 8 (Thermo Fisher Scientific, Waltham, MA, USA, 11-9938-82). ..

    Immunofluorescence:

    Article Title: The glycolytic enzyme PFKFB3 alleviates DNA damage and chondrocyte senescence in osteoarthritis
    Article Snippet: Secondary antibody, goat anti-rabbit (Yeasen, 33101ES60; dilution 1:200), was incubated for 1 h. Images were taken using a Digital Pathology Scanner (Leica, Wetzlar, Germany). .. In immunofluorescence, after antigen retrieval, primary antibodies against γH2AX (Abcam, ab181861; dilution 1:100), PFKFB3 (Cell Signaling Technology, D7H4Q; dilution 1:50), p16 INK4a (Cell Signaling Technology, #29271; 1:100), p21 (Santa Cruz, sc-397; 1:50), p-ATM (Cell Signaling Technology, #4526; dilution 1:100), p-ATR (Cell Signaling Technology, #30632; dilution 1:100) were incubated overnight. .. Then, slides were incubated with Alexa Fluor 488-conjugated secondary antibodies (Jackson ImmunoResearch, 111-545-003; dilution 1:500) for 1 h and 4′,6-diamidino-2-phenylindole (DAPI; Beyotime) for another 10 min at room temperature.

    Western Blot:

    Article Title: NSMF modulates replication stress to facilitate colorectal cancer progression
    Article Snippet: .. The following primary antibodies were used for western blot Antibodies against NSMF (Aviva System Biology, Cat# OAAN03468), γH2AX (Cell Signaling Technology, Cat# 9718), β-Actin (GeneTex, Cat# GTX629630), GAPDH (Santa Cruz, Cat# sc-32233), α-Tubulin (GeneTex, Cat# GTX112141), H2B (Millipore, Cat# 07-371), pT1989-ATR (Cell Signaling Technology, Cat# 30632), ATR (Santa Cruz, Cat# sc-515173), pS345-CHK1 (Cell Signaling Technology, Cat# 2348), pS317-CHK1 (Cell Signaling Technology, Cat# 2344), CHK1 (Santa Cruz, Cat# sc-8408), pS33-RPA2 (Bethyl Laboratories Inc., Cat# A300-246A), RPA2 (Bethyl Laboratories Inc., Cat# A300-244A), p16^INK4A (Cell Signaling Technology, Cat# 80 772), p21 Waf1/Cip1 (Cell Signaling Technology, Cat# 2947), Ras (Cell Signaling Technology, Cat# 14412), Cyclin D1 (Cell Signaling Technology, Cat# 2978), PARP (Cell Signaling Technology, Cat# 9532), Cleaved PARP (Cell Signaling Technology, Cat# 9541), Caspase 3 (Cell Signaling Technology, Cat# 9662), and Cleaved Caspase 3 (Cell Signaling Technology, Cat# 9661). .. Antibodies against Ki-67 (Abcam, Cat# 16667), Cleaved Caspase 3 (Cell Signaling Technology, Cat# 9661), and γH2AX (Cell Signaling Technology, Cat# 9718) were used for immunohistochemical staining.



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    Image Search Results


    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.

    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

    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.

    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

    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.

    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

    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.

    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

    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

    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

    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

    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

    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).

    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

    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).

    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

    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).

    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

    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).

    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