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: Purinergic Signalling

Article Title: Tumor growth inhibitory activity of the P2X7 receptor antagonist AZ10606120 in two cell lines of human glioblastoma

doi: 10.1007/s11302-026-10144-8

Figure Lengend Snippet: Newly synthesized cells are assessed by the BrdU proliferation assay after 72 h of treatment with AZ10606120 (5–100 µM). Panel A show results in U87MG cell line and Panel B show results in T98G cell line. Data are expressed as the percentage of AZ10606120-treated cells capable of incorporating BrdU compared to untreated control cells. Values ​​represent the mean ± standard error (SEM) of three independent experiments performed in six replicates and evaluated by one-way ANOVA followed by Dunnett's post-test. ( N = 3) *** p < 0.005; **** p < 0.0001. All p-values ​​were calculated relative to the control

Article Snippet: Cell proliferation was measured by bromodeoxyuridine (BrdU) incorporation using the BrdU Cell Proliferation Assay Kit purchased from Cell Signaling (Cat. No.:6813).

Techniques: Synthesized, Proliferation Assay, Control

Journal: bioRxiv

Article Title: STING causes replication stress and nascent DNA degradation via SAMHD1

doi: 10.64898/2026.03.28.714577

Figure Lengend Snippet: ( A ) Representative images of PLA on HDF shows STING-Lamin A interaction (foci) in control and progerin (PG)-expressing cells. DAPI is shown labeling genomic DNA. ( B ) Immunofluorescence analysis of BrdU incorporation in HDF treated with vehicle or doxycycline (Doxy) for 4 or 8 days to induce GFP-progerin, with or without STING inhibitor H151. ( C ) BrdU incorporation in HDF and progerin-expressing HDF transfected with STING siRNA (siSTING) or non-targeting control (siSCR). ( D ) Immunoblot analysis of STING, GFP-progerin, and phosphorylated RPA ( S33 p-RPA) in HDF control and progerin-expressing HDF in which STING is abrogated (H151 or siSTING). ( E ) IF images showing S33 p-RPA foci (red) in cells treated with Doxy ± H151 for 4 or 8 days; DAPI (blue) labels nuclei and white arrows indicate cytosolic foci. Scale bars, 20 µm ( F ) Quantification of cells with ≥3 nuclear S33 p-RPA foci per nucleus from (E). ( G ) Quantification of cells with nuclear S33 p-RPA foci in siSTING-or control siSCR-transfected cells treated with Doxy for 8 days. ( H ) Percentage of cells showing with ≥3 cytosolic S33 p-RPA foci from (E). ( I ) Cytosolic S33 p-RPA foci quantification in siSTING versus siSCR-transfected cells.

Article Snippet: Immunofluorescence was performed to analyze STING localization and activation (STING 1:500-Cell Signaling- 13647, S366 pSTING 1:1200- Cell Signaling-50907), BrdU quantification (1:200- Santa Cruz -sc20045), levels of S33 p-RPA (1:1000- Bethyl -PLA0070), γH2AX (1:200 Cell Signalling-2577) and 53BP1 (1:1000- Santa Cruz- sc517281).

Techniques: Control, Expressing, Labeling, Immunofluorescence, BrdU Incorporation Assay, Transfection, Western Blot