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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 <t>p16</t> 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).

P16 Ink4a Antibody F 12, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 96/100, based on 2157 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "The Flip Side of the Coin: METTL3 Serves as a Novel Cellular Senescence Accelerator via Negative Regulation of ITGA9"

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

Journal: Aging and Disease

doi: 10.14336/AD.2024.1715

Figure Legend 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).

Techniques Used: 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).

Figure Legend 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).

Techniques Used: 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).

Figure Legend 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).

Techniques Used: 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).

Figure Legend 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).

Techniques Used: Transfection, Infection, Staining, BrdU Incorporation Assay

Image Search Results

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).

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).

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).

Techniques: Transfection, Infection, Staining, BrdU Incorporation Assay

Nob decreased the expression of senescence markers in D-gal-induced senescent cells. ( A , B ) Representative images of SA-β-gal staining in CK and D-gal-induced C2C12 myoblasts treated for 48 h with DMSO or Nob. Scale bar, 200 μm. ( C , D ) Immunostaining of P16 in CK and D-gal-induced C2C12 myoblasts treated for 48 h with DMSO or Nob. Representative images are shown. Scale bar, 25 μm. ( E ) mRNA of P53 and P21 in CK and D-gal-induced C2C12 myoblasts treated for 48 h with DMSO or Nob. ( F ) Western blot analysis of P16, P53, P21, and GAPDH in CK and D-gal-induced C2C12 myoblasts treated for 48 h with DMSO or Nob. Different lowercase letters represent significant differences between different treatment groups ( p < 0.05).

Journal: International Journal of Molecular Sciences

Article Title: Nobiletin Prevents D-Galactose-Induced C2C12 Cell Aging by Improving Mitochondrial Function

doi: 10.3390/ijms231911963

Figure Lengend Snippet: Nob decreased the expression of senescence markers in D-gal-induced senescent cells. ( A , B ) Representative images of SA-β-gal staining in CK and D-gal-induced C2C12 myoblasts treated for 48 h with DMSO or Nob. Scale bar, 200 μm. ( C , D ) Immunostaining of P16 in CK and D-gal-induced C2C12 myoblasts treated for 48 h with DMSO or Nob. Representative images are shown. Scale bar, 25 μm. ( E ) mRNA of P53 and P21 in CK and D-gal-induced C2C12 myoblasts treated for 48 h with DMSO or Nob. ( F ) Western blot analysis of P16, P53, P21, and GAPDH in CK and D-gal-induced C2C12 myoblasts treated for 48 h with DMSO or Nob. Different lowercase letters represent significant differences between different treatment groups ( p < 0.05).

Article Snippet: Antibodies used were p16 INK4A (F-12) (Santa Cruz Biotechnology, sc-1661), P53 mouse monoclonal antibody (Proteintech, 60283-2-Ig), P21 rabbit polyclonal antibody (Proteintech, 27296-1-AP), LC3B antibody (Cell Signaling, 2575S), LAMP2 (Abcam, ab13524), AMPK (Cell Signaling, D63G4), P-AMPK (Cell Signaling, 2535S), p70 S6 Kinase Antibody (Cell Signaling, 9202S), p-p70 S6 Kinase Antibody (Cell Signaling, 9234S), and GAPDH polyclonal antibody (Proteintech, 10494-1-AP).

Techniques: Expressing, Staining, Immunostaining, Western Blot

(A: upper panel) The levels of expression of the p16 Ink4a gene are significantly higher throughout the lifespan of WT mice compared to corresponding age matched DN-p38α AF/+ mice. Data for WT mice are depicted in (A1) young (2.0 mos); (A3) middle aged (13.5 mos); (A5) aged (20 mos); (A: lower panel) Data for DN-p38α AF/+ mice are depicted in (A2) young (2 mos); (A4) middle aged (13.5 mos) and aged (A6) aged (20 mos). (B) Bar graph presentation of the Western blot analyses of (C). *p = 0.05 vs. WT of the same age. (C) Western blot analyses of the level of expression of p16 Ink4a in gastrocnemius of young (2 mos); middle aged (13.5 mos) and aged (20 mos) WT and DN-p38α AF/+ mice.

Journal: Aging (Albany NY)

Article Title: Attenuation of p38α MAPK stress response signaling delays the in vivo aging of skeletal muscle myofibers and progenitor cells

doi:

Figure Lengend Snippet: (A: upper panel) The levels of expression of the p16 Ink4a gene are significantly higher throughout the lifespan of WT mice compared to corresponding age matched DN-p38α AF/+ mice. Data for WT mice are depicted in (A1) young (2.0 mos); (A3) middle aged (13.5 mos); (A5) aged (20 mos); (A: lower panel) Data for DN-p38α AF/+ mice are depicted in (A2) young (2 mos); (A4) middle aged (13.5 mos) and aged (A6) aged (20 mos). (B) Bar graph presentation of the Western blot analyses of (C). *p = 0.05 vs. WT of the same age. (C) Western blot analyses of the level of expression of p16 Ink4a in gastrocnemius of young (2 mos); middle aged (13.5 mos) and aged (20 mos) WT and DN-p38α AF/+ mice.

Article Snippet: The following primary antibodies used for immunofluorescence were purchased from Abcam, Cambridge, MA: beta actin, GAPDH, rabbit anti-ALDH1A1 (1:100, #ab52492,); rabbit anti-ALDH2 (1:200 #ab 108306); mouse anti-BubR1 (1:200, #ab54894); ); rabbit anti-p38α (1:1000, #ab7952); rabbit anti-COX2 (1:200, #ab15191); rabbit anti-protein-HNE (1:500, #HNE11-S, Alpha Diagnostic International, San Antonio, TX); rabbit anti-iNOS (1:500, #ab15323); mouse anti-p16 Ink4a (F-12) (1:200, #sc-1661, Santa Cruz Biotech, Dallas, TX); rabbit anti-p19 Arf (M-167) (1:200, #sc-1063, Santa Cruz Biotech); and rabbit anti-phosph-p38αMAPK (Thr180/Tyr182) (D3F9) (1:250, #4511, Cell Signaling Technology, Danvers, MA).

Techniques: Expressing, Western Blot

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