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

Journal: Nature metabolism

Article Title: BMP4 and Gremlin 1 regulate hepatic cell senescence during clinical progression of NAFLD/NASH.

doi: 10.1038/s42255-022-00620-x

Figure Lengend Snippet: Fig. 5 | BMP4 prevents the increase in DOX-induced senescence markers, whereas GREM1 enhances the effects of DOX. a,c, Representative immunoblots of respective proteins in control and DOX-treated cells stimulated with or without BMP4 (20 ng ml−1 and 50 ng ml−1) (a) or GREM1 (200 ng ml−1) (c) (n = 6 or 7 biologically independent experiments, except for LATS2 (n = 5) and pSMA1/5/9 (n = 4)). b, Representative immunofluorescent images of control and DOX-treated cells, stimulated with or without BMP4 or GREM1, stained for p53 (green), p16 (green) and nuclei (DAPI, blue). Scale bars represent 20 µm. Bar graph displays fluorescence intensities quantified using ImageJ and normalized to the number of nuclei (n = 3, 6–10 randomly chosen fields from each experiment). Values are mean ± s.e.m. Statistics were calculated using one-way ANOVA followed by Bonferroni’s post hoc test.

Article Snippet: The following antibodies were used: For WB: p53 (7F5) (Cell Signaling Technology; 2527; 1:1000) p16 INK4A (E6N8P) (Cell Signaling Technology; 18769; 1:1000) p21 Waf1/Cip1/CDKN1A Antibody (F-5) (Santa Cruz Biotechnology; sc-6246; 1:1000) Phospho-Histone H2A.X (Ser139) (D7T2V) (Cell Signaling Technology; 80312; 1:1000) -Galactosidase (E2U2I) (Cell Sinaling Technology; 27198; 1:1000) Phospho-MDM2 (Ser166) (Cell Signaling Technology; 3521; 1:1000) MDM2 (D1V2Z) (Cell Signaling Technology; 86934; 1:1000) Cleaved Caspase-3 (Asp175) (5A1E) (Cell Signaling Technology; 9664; 1:1000) LATS1 (C66B5) (Cell Signaling Technology; 3477; 1:1000) LATS2 (D83D6) (Cell Signaling Technology; 5888; 1:1000) Phospho-YAP (Ser127) (D9W2I) (Cell Signaling Technology; 13008; 1:1000) Phospho-TAZ (Ser89) (E1X9C) (Cell Signaling Technology; 59971; 1:1000)

Techniques: Western Blot, Control, Staining, Fluorescence