Journal: JCI Insight

Article Title: GLS1 governs vascular smooth muscle cell phenotypic switching and aortic dissection via glutamate metabolism

doi: 10.1172/jci.insight.203575

Figure Lengend Snippet: ( A ) Human aortic smooth muscle cells (HASMCs) were treated with PDGF-BB. Western blot analysis of GLS1 and TAGLN expression. ( B and C ) HASMCs were transfected with siRNA against GLS1 (si GLS1 ) or negative control (siNC), and then treated with PDGF-BB. ( B ) The mRNA expression of GLS1 , TAGLN , ACTA2 , OPN , MMP2 , and MMP9 in HASMCs was detected by real-time qPCR. ( C ) Immunofluorescence images of in situ zymography (DQ gelatin) in HASMCs. MMP activity (green) was quantified by immunofluorescence intensity. Scale bars: 50 μm. (Blue, DAPI.) Data are presented as mean ± SEM. Statistical analysis was performed using unpaired, 2-tailed Student’s t test ( A ), or 1-way ANOVA followed by Tukey’s multiple-comparison test ( B and C ). * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: For stimulation, HASMCs were serum-starved in DMEM without FBS for 12 hours, followed by stimulation with PDGF-BB (MedChemExpress, catalog HY-P7055) at the indicated concentration (20 ng/mL) for 24 hours. siRNAs for GLS1 and RARα (GenePharma) were transfected using Lipofectamine 3000 (Invitrogen, catalog L3000075), with scrambled siRNA as negative control.

Techniques: Western Blot, Expressing, Transfection, Negative Control, Immunofluorescence, In Situ, Zymography, Activity Assay, Comparison

Journal: JCI Insight

Article Title: GLS1 governs vascular smooth muscle cell phenotypic switching and aortic dissection via glutamate metabolism

doi: 10.1172/jci.insight.203575

Figure Lengend Snippet: ( A and B ) HASMCs were infected with adenovirus containing empty vector (ad-Vector) or GLS1-encoding plasmids (ad- GLS1 ), and then treated with PDGF-BB. ( A ) The mRNA expression of GLS1 , TAGLN , ACTA2 , OPN , MMP2 , and MMP9 in HASMCs was detected by real-time qPCR. ( B ) Immunofluorescence images of in situ zymography (DQ gelatin) in HASMCs. MMP activity (green) was quantified by immunofluorescence intensity. Scale bars: 50 μm. (Blue: DAPI.) Data are presented as mean ± SEM. Statistical analysis was performed using 1-way ANOVA followed by Tukey’s multiple-comparison test ( A and B ). ** P < 0.01, *** P < 0.001.

Article Snippet: For stimulation, HASMCs were serum-starved in DMEM without FBS for 12 hours, followed by stimulation with PDGF-BB (MedChemExpress, catalog HY-P7055) at the indicated concentration (20 ng/mL) for 24 hours. siRNAs for GLS1 and RARα (GenePharma) were transfected using Lipofectamine 3000 (Invitrogen, catalog L3000075), with scrambled siRNA as negative control.

Techniques: Infection, Plasmid Preparation, Expressing, Immunofluorescence, In Situ, Zymography, Activity Assay, Comparison

Journal: JCI Insight

Article Title: GLS1 governs vascular smooth muscle cell phenotypic switching and aortic dissection via glutamate metabolism

doi: 10.1172/jci.insight.203575

Figure Lengend Snippet: ( A ) RNA sequencing was performed on HASMCs infected with adenovirus containing empty vector (ad-Vector) or GLS1-encoding plasmids (ad- GLS1 ) and then treated with PDGF-BB. Venn diagram showing the overlap DEGs between ad-Vector+PDGF-BB versus ad-Vector and ad- GLS1 +PDGF-BB versus ad-Vector+PDGF-BB ( n = 3). ( B ) KEGG enrichment analysis of these overlap DEGs. ( C ) Schematic diagram of GLS1-related metabolic pathway for glutamine. ( D – F ) HASMCs were infected with adenovirus containing empty vector (ad-Vector) or GLS1-encoding plasmids (ad- GLS1 ), and then treated with PDGF-BB. ( D ) The content of glutamate in HASMCs. ( E ) The content of glutathione (GSH) in HASMCs. ( F ) The content of α-ketoglutarate (αKG) in HASMCs. Data are presented as mean ± SEM. Statistical analysis was performed using 1-way ANOVA followed by Tukey’s multiple-comparison test ( D – F ). * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: For stimulation, HASMCs were serum-starved in DMEM without FBS for 12 hours, followed by stimulation with PDGF-BB (MedChemExpress, catalog HY-P7055) at the indicated concentration (20 ng/mL) for 24 hours. siRNAs for GLS1 and RARα (GenePharma) were transfected using Lipofectamine 3000 (Invitrogen, catalog L3000075), with scrambled siRNA as negative control.

Techniques: RNA Sequencing, Infection, Plasmid Preparation, Comparison

Journal: JCI Insight

Article Title: GLS1 governs vascular smooth muscle cell phenotypic switching and aortic dissection via glutamate metabolism

doi: 10.1172/jci.insight.203575

Figure Lengend Snippet: ( A – C ) HASMCs were infected with adenovirus containing empty vector (ad-Vector) or GLS1-encoding plasmids (ad- GLS1 ), and then treated with PDGF-BB. ( A ) Summarized oxygen consumption rate (OCR) tracings in HASMCs. Basal OCR was measured, followed by sequential injections of 1 μM oligomycin, 1.5 μM FCCP, and a mixture of 2 μM rotenone and 2 μM antimycin A. ( B ) Mitochondrial reactive oxygen species were detected by MitoSOX staining in HASMCs. Scale bar: 50 μm. ( C ) Western blot analysis of phosphorylated and total PI3K, AKT, and mTOR levels in HASMCs. Data are presented as mean ± SEM. Statistical analysis was performed using 1-way ANOVA followed by Tukey’s multiple-comparison test ( B and C ). * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: For stimulation, HASMCs were serum-starved in DMEM without FBS for 12 hours, followed by stimulation with PDGF-BB (MedChemExpress, catalog HY-P7055) at the indicated concentration (20 ng/mL) for 24 hours. siRNAs for GLS1 and RARα (GenePharma) were transfected using Lipofectamine 3000 (Invitrogen, catalog L3000075), with scrambled siRNA as negative control.

Techniques: Infection, Plasmid Preparation, Staining, Western Blot, Comparison

Journal: JCI Insight

Article Title: GLS1 governs vascular smooth muscle cell phenotypic switching and aortic dissection via glutamate metabolism

doi: 10.1172/jci.insight.203575

Figure Lengend Snippet: ( A ) Prediction of GLS1 promoter–binding transcription factors by AnimalTFDB and using the region from 2,000 bp upstream to 100 bp downstream of the GLS1 transcription start site (TSS) as the promoter region. Venn diagram of DEGs in human aortic dissection samples relative to normal controls from the NCBI GEO database ( GSE52093 and GSE98770 ). ( B ) Western blot analysis of RARα expression in the aorta from non-AD groups and AD patients. ( C ) The construction of wild-type (WT) and 3 mutated luciferase reporter gene plasmids for GLS1 promoter by mutation of 3 potential binding sites of RARα predicted by JASPAR. ( D ) Relative luciferase activity in HEK293 cells of luciferase reporter constructs containing GLS1 promoter or its mutants transfected along with pRL-TK (internal control plasmid) followed by transfection with RARα-encoding plasmid. ( E and F ) HASMCs were transfected with siRNA against RAR α (si RAR α ) or negative control (siNC), and then treated with PDGF-BB. ( E ) The mRNA expression of RAR α , GLS1 , TAGLN , ACTA2 , and OPN in HASMCs was detected by real-time qPCR. ( F ) Immunofluorescence images of in situ zymography (DQ gelatin) in HASMCs. MMP activity (green) was quantified by immunofluorescence intensity. Scale bars: 50 μm. (Blue: DAPI.) Data are presented as mean ± SEM. Statistical analysis was performed using unpaired, 2-tailed Student’s t test ( B ), or 1-way ANOVA followed by Tukey’s multiple-comparison test ( D – F ). * P < 0.05, *** P < 0.001.

Article Snippet: For stimulation, HASMCs were serum-starved in DMEM without FBS for 12 hours, followed by stimulation with PDGF-BB (MedChemExpress, catalog HY-P7055) at the indicated concentration (20 ng/mL) for 24 hours. siRNAs for GLS1 and RARα (GenePharma) were transfected using Lipofectamine 3000 (Invitrogen, catalog L3000075), with scrambled siRNA as negative control.

Techniques: Binding Assay, Dissection, Western Blot, Expressing, Luciferase, Mutagenesis, Activity Assay, Construct, Transfection, Control, Plasmid Preparation, Negative Control, Immunofluorescence, In Situ, Zymography, Comparison