Journal: Cell Death Discovery

Article Title: N -Acetylneuraminic acid triggers endothelial pyroptosis and promotes atherosclerosis progression via GLS2-mediated glutaminolysis pathway

doi: 10.1038/s41420-024-02233-7

Figure Lengend Snippet: A , B ECs were treated with the indicated concentrations (0, 5, 10, 15, 20, 25, 30 and 40 mM) of Neu5Ac for 12 h. The cytotoxicity was measured by using a CCK-8 detection kit. C The morphology of EA.hy926 cells treated with 20 mM Neu5Ac in vitro for 12 or 24 h under optical microscopy. Bar = 100 µm. D The morphology of EA.hy926 cells treated with 20 mM Neu5Ac for 12 h under transmission electron microscopy. Bar = 1 µm. E , F Western blot analysis of pyroptosis markers (GSDMD-N, GSDME, NLRP3 inflammasome, Caspase-1, IL-1β, and IL-18) and pro-inflammatory adhesion molecules (ICAM-1 and VCAM-1) protein expression in ECs treated with indicated concentrations of Neu5Ac for 12 h. G , H qRT-PCR analysis of IL-1β, IL-6, Caspase-1, NLRP3 and ICAM-1 expression in ECs treated with indicated concentrations of Neu5Ac for 12 h. I , J Pore formation in the cell membrane of HUVECs treated with indicated concentrations of Neu5Ac for 12 h was observed by Hoechst/PI staining. Bar = 100 μm. K , L ECs were preincubated with or without Z-VAD-FMK (10 µM) for 1 h before Neu5Ac (20 mM) treatment for 12 h, IL-1β, IL-18, Caspase-1, NLRP3, GSDMD-N and ICAM-1 protein expression were assessed in the ECs by western blot. M , N ECs were preincubated with or without MCC950 (10 µM) for 1 h before Neu5Ac (20 mM) treatment for 12 h, IL-1β, IL-18, Caspase-1, NLRP3, GSDMD-N and ICAM-1 protein expression were assessed in the ECs by western blot. Data were analyzed using unpaired two-tailed Student’s t -tests or one-way ANOVA tests, and presented as the means ± SEM. * p < 0.05 was considered significant, ** p < 0.01, *** p < 0.001.

Article Snippet: The primary antibodies used for immunoblots were as follows: Rabbit Polyclonal Antibody against IL-1β (Beyotime, AF7209, diluted 1:1000), Rabbit Monoclonal Antibody against NLRP3 (Beyotime, AF2155, diluted 1:1000), Rabbit Monoclonal Antibody against Caspase-1 (Beyotime, AF1681, diluted 1:1000), Rabbit Monoclonal Antibody against VCAM-1 (Beyotime, AF1021, diluted 1:1000), Rabbit Polyclonal Antibody against ICAM-1 (Beyotime, AF0195, diluted 1:1000), Rabbit Polyclonal Antibody against GLS2 (ABclonal, A16029, diluted 1:1000), Rabbit Polyclonal Antibody against GSDMD (full length + N-terminal) (ABclonal, A20197, diluted 1:1000), Rabbit Polyclonal Antibody against GSDME (ABclonal, A7432, diluted 1:1000), Rabbit Polyclonal Antibody against IL-18 (ABclonal, A16737, diluted 1:1000), Rabbit Polyclonal Antibody against Phospho-FOXO3A-S253 (ABclonal, AP0684, diluted 1:1000), Rabbit Polyclonal Antibody against c-Myc (Proteintech, 10828-1-AP, diluted 1:1000), mouse Monoclonal Antibody against FOXO3a (Proteintech, 66428-1-Ig, diluted 1:1000), mouse Monoclonal Antibody against SIRT3 (Santa Cruz, sc-365175, diluted 1:1000).

Techniques: CCK-8 Assay, In Vitro, Microscopy, Transmission Assay, Electron Microscopy, Western Blot, Expressing, Quantitative RT-PCR, Membrane, Staining, Two Tailed Test

Journal: Cell Death Discovery

Article Title: N -Acetylneuraminic acid triggers endothelial pyroptosis and promotes atherosclerosis progression via GLS2-mediated glutaminolysis pathway

doi: 10.1038/s41420-024-02233-7

Figure Lengend Snippet: A DHE staining showed intracellular superoxide production in aortic root from 0.9% N.S. ( n = 3) or Neu5Ac-injected ApoE −/− mice ( n = 3). Bar = 500 µm. B Electron microscopy of the mitochondria of Neu5Ac-treated EA.hy926 cells. Bar = 1 µm. C EA.hy926 cells were treated with indicated concentrations of Neu5Ac for 12 h, mitochondrial mass was labeled by MitoTracker Green; the mitochondrial membrane potential was assayed by TMRE; the mitochondrial ROS level was detected by mitoSOX. Bar = 31.75 µm. D The intracellular ROS were measured by DCFH-DA probe in Neu5Ac-treated EA.hy926 cells. EA.hy926 cells were preincubated with or without ROS scavenger NAC (5 mM) for 1 h before Neu5Ac (20 mM) treatment for 12 h, IL-1β, IL-18, Caspase-1, NLRP3, GSDMD-N and ICAM-1 protein expression were assessed by western blot, with quantitative data at right ( E , F ); and cell death in HUVEC was detected by PI staining, with quantitative data at below ( G , H ). Bar = 100 µm. Data were analyzed using unpaired two-tailed Student’s t -tests or one-way ANOVA tests, and presented as the means ± SEM. * p < 0.05 was considered significant, ** p < 0.01, *** p < 0.001.

Article Snippet: The primary antibodies used for immunoblots were as follows: Rabbit Polyclonal Antibody against IL-1β (Beyotime, AF7209, diluted 1:1000), Rabbit Monoclonal Antibody against NLRP3 (Beyotime, AF2155, diluted 1:1000), Rabbit Monoclonal Antibody against Caspase-1 (Beyotime, AF1681, diluted 1:1000), Rabbit Monoclonal Antibody against VCAM-1 (Beyotime, AF1021, diluted 1:1000), Rabbit Polyclonal Antibody against ICAM-1 (Beyotime, AF0195, diluted 1:1000), Rabbit Polyclonal Antibody against GLS2 (ABclonal, A16029, diluted 1:1000), Rabbit Polyclonal Antibody against GSDMD (full length + N-terminal) (ABclonal, A20197, diluted 1:1000), Rabbit Polyclonal Antibody against GSDME (ABclonal, A7432, diluted 1:1000), Rabbit Polyclonal Antibody against IL-18 (ABclonal, A16737, diluted 1:1000), Rabbit Polyclonal Antibody against Phospho-FOXO3A-S253 (ABclonal, AP0684, diluted 1:1000), Rabbit Polyclonal Antibody against c-Myc (Proteintech, 10828-1-AP, diluted 1:1000), mouse Monoclonal Antibody against FOXO3a (Proteintech, 66428-1-Ig, diluted 1:1000), mouse Monoclonal Antibody against SIRT3 (Santa Cruz, sc-365175, diluted 1:1000).

Techniques: Staining, Injection, Electron Microscopy, Labeling, Membrane, Expressing, Western Blot, Two Tailed Test

Journal: Cell Death Discovery

Article Title: N -Acetylneuraminic acid triggers endothelial pyroptosis and promotes atherosclerosis progression via GLS2-mediated glutaminolysis pathway

doi: 10.1038/s41420-024-02233-7

Figure Lengend Snippet: A qRT-PCR analysis of key enzymes of glutaminolysis process (GLS, GLSiso1, GLSiso2 and GLS2) in EA.hy926 cells treated with indicated concentrations of Neu5Ac (0, 5, 10 and 20 mM) for 12 h. B , C EA.hy926 cells were treated with Neu5Ac (20 mM) for 12 h, GLS2 expression was assessed by western blot, with quantitative data at below. D CD31 and GLS2 immunostaining of aortic root from 0.9% N.S. ( n = 3) or Neu5Ac-injected ApoE − /− mice ( n = 3), with quantitative data at below. Bar = 100 µm. E qRT-PCR analysis of GLS2 in aortic artery from 0.9% N.S. ( n = 3) or Neu5Ac-injected ApoE − /− mice ( n = 3). EA.hy926 cells were preincubated with or without Compound 968 (10 µM) for 1 h or transfected with GLS2 siRNA before Neu5Ac (20 mM) treatment for 12 h, mitochondrial mass, membrane potential and mitochondrial ROS production were labeled, bar = 31.75 µm ( F , G ); IL-1β, IL-18, Caspase-1, NLRP3, GSDMD-N and ICAM-1 protein expression were assessed by western blot ( H , I ). EA.hy926 cells were preincubated with or without Compound 968 (10 µM) for 1 h before Neu5Ac (20 mM) treatment for 12 h, the intracellular level of glutamate ( J ) and glutamine ( K ) were assessed by ELISA; the intracellular level of GSH was measured by using Glutathione Assay Kit ( L ); the intracellular level of α-KG was measured by LC/MS ( M ). N Western blot analysis of IL-1β, IL-18, Caspase-1, NLRP3, GSDMD and ICAM-1 of EA.hy926 cells deprived of Gln (-Gln) or transfected with GLS2 siRNA followed by Neu5Ac (20 mM) treatment for 12 h in the presence or absence of 4 mM DMKG for 3 h. Data were analyzed using unpaired two-tailed Student’s t -tests or one-way ANOVA tests, and presented as the means ± SEM. * p < 0.05 was considered significant, ** p < 0.01, *** p < 0.001.

Article Snippet: The primary antibodies used for immunoblots were as follows: Rabbit Polyclonal Antibody against IL-1β (Beyotime, AF7209, diluted 1:1000), Rabbit Monoclonal Antibody against NLRP3 (Beyotime, AF2155, diluted 1:1000), Rabbit Monoclonal Antibody against Caspase-1 (Beyotime, AF1681, diluted 1:1000), Rabbit Monoclonal Antibody against VCAM-1 (Beyotime, AF1021, diluted 1:1000), Rabbit Polyclonal Antibody against ICAM-1 (Beyotime, AF0195, diluted 1:1000), Rabbit Polyclonal Antibody against GLS2 (ABclonal, A16029, diluted 1:1000), Rabbit Polyclonal Antibody against GSDMD (full length + N-terminal) (ABclonal, A20197, diluted 1:1000), Rabbit Polyclonal Antibody against GSDME (ABclonal, A7432, diluted 1:1000), Rabbit Polyclonal Antibody against IL-18 (ABclonal, A16737, diluted 1:1000), Rabbit Polyclonal Antibody against Phospho-FOXO3A-S253 (ABclonal, AP0684, diluted 1:1000), Rabbit Polyclonal Antibody against c-Myc (Proteintech, 10828-1-AP, diluted 1:1000), mouse Monoclonal Antibody against FOXO3a (Proteintech, 66428-1-Ig, diluted 1:1000), mouse Monoclonal Antibody against SIRT3 (Santa Cruz, sc-365175, diluted 1:1000).

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Immunostaining, Injection, Transfection, Membrane, Labeling, Enzyme-linked Immunosorbent Assay, Glutathione Assay, Liquid Chromatography with Mass Spectroscopy, Two Tailed Test

Journal: Cell Death Discovery

Article Title: N -Acetylneuraminic acid triggers endothelial pyroptosis and promotes atherosclerosis progression via GLS2-mediated glutaminolysis pathway

doi: 10.1038/s41420-024-02233-7

Figure Lengend Snippet: A Diagram showing generation and pathologic analysis of HFD, GLS2 KD, OP-administrated ApoE −/− mice ( n ≥ 3). Oil Red O staining and quantitative data of atherosclerotic lesions of aorta en face ( B , G ) and aortic root ( C , H ) in WT, HFD, GLS2 KD and OP-administrated ApoE −/− mice. Bar = 100 µm. HE staining ( D , I ), Masson staining ( E , J ) and DHE staining ( F , K ) of atherosclerotic lesions of aortic root, with quantitative data right. L , M qRT-PCR analysis of IL-1β and IL-6 expression in aortic artery from WT, HFD, GLS2 KD and OP-administrated ApoE −/− mice. Data were analyzed using unpaired two-tailed Student’s t -tests or one-way ANOVA tests, and presented as the means ± SEM. * p < 0.05 was considered significant, ** p < 0.01, *** p < 0.001.

Article Snippet: The primary antibodies used for immunoblots were as follows: Rabbit Polyclonal Antibody against IL-1β (Beyotime, AF7209, diluted 1:1000), Rabbit Monoclonal Antibody against NLRP3 (Beyotime, AF2155, diluted 1:1000), Rabbit Monoclonal Antibody against Caspase-1 (Beyotime, AF1681, diluted 1:1000), Rabbit Monoclonal Antibody against VCAM-1 (Beyotime, AF1021, diluted 1:1000), Rabbit Polyclonal Antibody against ICAM-1 (Beyotime, AF0195, diluted 1:1000), Rabbit Polyclonal Antibody against GLS2 (ABclonal, A16029, diluted 1:1000), Rabbit Polyclonal Antibody against GSDMD (full length + N-terminal) (ABclonal, A20197, diluted 1:1000), Rabbit Polyclonal Antibody against GSDME (ABclonal, A7432, diluted 1:1000), Rabbit Polyclonal Antibody against IL-18 (ABclonal, A16737, diluted 1:1000), Rabbit Polyclonal Antibody against Phospho-FOXO3A-S253 (ABclonal, AP0684, diluted 1:1000), Rabbit Polyclonal Antibody against c-Myc (Proteintech, 10828-1-AP, diluted 1:1000), mouse Monoclonal Antibody against FOXO3a (Proteintech, 66428-1-Ig, diluted 1:1000), mouse Monoclonal Antibody against SIRT3 (Santa Cruz, sc-365175, diluted 1:1000).

Techniques: Staining, Quantitative RT-PCR, Expressing, Two Tailed Test

Journal: Cell Death Discovery

Article Title: N -Acetylneuraminic acid triggers endothelial pyroptosis and promotes atherosclerosis progression via GLS2-mediated glutaminolysis pathway

doi: 10.1038/s41420-024-02233-7

Figure Lengend Snippet: EA.hy926 cells were preincubated with or without 10058-F4 (60 µM) for 1 h before Neu5Ac (20 mM) treatment for 12 h, GLS2 and DAPI immunostaining were performed, with quantitative data at below; bar = 100 µm ( A , B ); IL-1β, IL-18, Caspase-1, NLRP3, GSDMD-N, GLS2 and ICAM-1 protein expression were assessed by western blot, with quantitative data at below ( C , D ); mitochondrial mass, membrane potential and mitochondrial ROS production were labeled by MitoTracker Green, TMRE and mitoSOX respectively; bar = 31.75 µm ( E ). EA.hy926 cells were transfected with SIRT3 siRNA or NC siRNA before Neu5Ac (20 mM) treatment for 12 h, IL-1β, IL-18, Caspase-1, NLRP3, GSDMD-N, GLS2, p-FOXO3a and ICAM-1 protein expression were assessed by western blot and quantified ( F , G ). Mitochondrial mass, membrane potential and mitochondrial ROS production were labeled by MitoTracker Green, TMRE and mitoSOX respectively; bar = 31.75 µm ( H ). I , J Western blot analysis of IL-1β, IL-18, Caspase-1, NLRP3, GSDMD-N and ICAM-1 of EA.hy926 cells transfected with GLS2 plasmid and SIRT3 siRNA followed by Neu5Ac (20 mM) treatment for 12 h. K EA.hy926 cells were transfected with SIRT3 siRNA or NC siRNA before Neu5Ac (20 mM) treatment for 12 h, Chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR) was performed with an antibody for c-Myc and antibody for IgG as the negative control. Data were analyzed using unpaired two-tailed Student’s t -tests or one-way ANOVA tests, and presented as the means ± SEM. * p < 0.05 was considered significant, ** p < 0.01, *** p < 0.001.

Article Snippet: The primary antibodies used for immunoblots were as follows: Rabbit Polyclonal Antibody against IL-1β (Beyotime, AF7209, diluted 1:1000), Rabbit Monoclonal Antibody against NLRP3 (Beyotime, AF2155, diluted 1:1000), Rabbit Monoclonal Antibody against Caspase-1 (Beyotime, AF1681, diluted 1:1000), Rabbit Monoclonal Antibody against VCAM-1 (Beyotime, AF1021, diluted 1:1000), Rabbit Polyclonal Antibody against ICAM-1 (Beyotime, AF0195, diluted 1:1000), Rabbit Polyclonal Antibody against GLS2 (ABclonal, A16029, diluted 1:1000), Rabbit Polyclonal Antibody against GSDMD (full length + N-terminal) (ABclonal, A20197, diluted 1:1000), Rabbit Polyclonal Antibody against GSDME (ABclonal, A7432, diluted 1:1000), Rabbit Polyclonal Antibody against IL-18 (ABclonal, A16737, diluted 1:1000), Rabbit Polyclonal Antibody against Phospho-FOXO3A-S253 (ABclonal, AP0684, diluted 1:1000), Rabbit Polyclonal Antibody against c-Myc (Proteintech, 10828-1-AP, diluted 1:1000), mouse Monoclonal Antibody against FOXO3a (Proteintech, 66428-1-Ig, diluted 1:1000), mouse Monoclonal Antibody against SIRT3 (Santa Cruz, sc-365175, diluted 1:1000).

Techniques: Immunostaining, Expressing, Western Blot, Membrane, Labeling, Transfection, Plasmid Preparation, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, ChIP-qPCR, Negative Control, Two Tailed Test