Journal: bioRxiv

Article Title: Metformin attenuates hyperglycaemia-stimulated pro-fibrotic gene expression in vascular adventitial fibroblasts via inhibition of Discoidin Domain Receptor 2

doi: 10.1101/2022.10.24.513528

Figure Lengend Snippet: Cells were pre-treated with metformin hydrochloride (10 μM), 45 mins prior to HG supplementation. (A) DDR2 mRNA levels were determined by Taqman real-time PCR at 6 h post-HG treatment, with β-actin as loading control. **p< 0.005 vs. control, ##p< 0.005 vs. HG (One-way ANOVA p< 0.05). (B) Protein was isolated after 12h of HG treatment and subjected to western blot analysis for detection of DDR2, with β-actin as loading control. **p< 0.005 vs. control, ##p< 0.005 vs. HG. Data are representative of 3 independent experiments, n=3. Error bars represent Standard Error (SE).

Article Snippet: Primary antibody against DDR2 (Cat No. 12133S) and TGF-β1 (Cat No. 3711) were obtained from Cell Signalling Technology (Danvers, MA, USA).

Techniques: Real-time Polymerase Chain Reaction, Control, Isolation, Western Blot

Journal: bioRxiv

Article Title: Metformin attenuates hyperglycaemia-stimulated pro-fibrotic gene expression in vascular adventitial fibroblasts via inhibition of Discoidin Domain Receptor 2

doi: 10.1101/2022.10.24.513528

Figure Lengend Snippet: Vascular adventitial fibroblasts were transiently transfected with DDR2 siRNA or scrambled siRNA. Following exposure of the transfected cells to HG for 12 h, fibronectin protein expression was examined by western blot analysis and normalized to β-actin. **p< 0.005 vs. control, #p< 0.05 vs. HG. Data are representative of 3 independent experiments, n=3. Error bars represent Standard Error (SE).

Article Snippet: Primary antibody against DDR2 (Cat No. 12133S) and TGF-β1 (Cat No. 3711) were obtained from Cell Signalling Technology (Danvers, MA, USA).

Techniques: Transfection, Expressing, Western Blot, Control

Journal: bioRxiv

Article Title: Metformin attenuates hyperglycaemia-stimulated pro-fibrotic gene expression in vascular adventitial fibroblasts via inhibition of Discoidin Domain Receptor 2

doi: 10.1101/2022.10.24.513528

Figure Lengend Snippet: (A) Vascular adventitial fibroblasts were transiently transfected with TGF-β1 siRNA 1 and 2 (5 pmol each) or scrambled siRNA prior to treatment with HG for 12 h. Protein was isolated and subjected to western blot analysis for detection of fibronectin, with β-actin as loading control. Validation of TGF-β1 knockdown is also shown. **p< 0.005 vs. control, ##p< 0.005 vs. HG and ns -not significant vs. control. (B) Sub-confluent, quiescent cultures of vascular adventitial fibroblasts in M199 were pre-treated with SMAD2 inhibitor, PD169316, or SMAD3 inhibitor, SIS3, for 1 h and subsequently with HG for 12 h. Following treatment with HG, protein was isolated and subjected to western blot analysis for detection of fibronectin, with β-actin as loading control. **p< 0.005 vs. control, ##p< 0.005 vs. HG and ns - not significant vs. control. (C) Vascular adventitial fibroblasts were co-transfected with DDR2 plasmid (1μg) and TGF-β1 siRNA 1 and 2 (5 pmol each) (lipofectamine 2000-complexed) for 8 h in OptiMEM. After a recovery period of 12 h, the cells were subjected to HG for 12 h. Following treatment with HG, protein was isolated and subjected to western blot analysis for detection of fibronectin, with β-actin as loading control. Validation of DDR2 OE and TGF-β1 knockdown is also shown. **p<0.005 vs. Control, ##p<0.005 vs. HG, !!p<0.005 vs. HG + TGF-β siRNA. Data are representative of 3 independent experiments, n=3. Error bars represent Standard Error (SE).

Article Snippet: Primary antibody against DDR2 (Cat No. 12133S) and TGF-β1 (Cat No. 3711) were obtained from Cell Signalling Technology (Danvers, MA, USA).

Techniques: Transfection, Isolation, Western Blot, Control, Biomarker Discovery, Knockdown, Plasmid Preparation