pgw1cmv perceval plasmid  (Addgene inc)

Journal: iScience

Article Title: The endoplasmic reticulum plays a key role in α-cell intracellular Ca 2+ dynamics and glucose-regulated glucagon secretion in mouse islets

doi: 10.1016/j.isci.2024.109665

Figure Lengend Snippet:

Article Snippet: GW1CMV-Perceval plasmid , Berg et al. , AddGene: #21737.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Software

Journal: Scientific Reports

Article Title: Inhibition of mitochondrial function by metformin increases glucose uptake, glycolysis and GDF-15 release from intestinal cells

doi: 10.1038/s41598-021-81349-7

Figure Lengend Snippet: Metformin increases glycolysis in intestinal cells. ( a ) Changes in extracellular acidification rate (ECAR) during the glycolytic stress test wherein intestinal cells were pre-treated with control or 1 mM metformin for 24 h. Results were normalised to the protein concentration measured by BCA assay. Dotted lines indicate when compound were added: Gluc glucose (10 mM), OliA oligomycin-A (1 μM), 2-DG 2-deoxyglucose (50 mM). Inset: Effect of metformin on measured parameters of the glycolysis stress test. n = 10 wells from 3 independent experiments. NS not significant. ***P < 0.001. Two-way ANOVA and Bonferroni post-hoc test. ( b ) Example traces of Perceval fluorescence ratios from Control and Metformin treated cells in response to 2-DG (50 mM), with violin plots shows of the change in fluorescent intensity ratio (FI). ***P < 0.001, Student’s t test. Control; n = 14 from 4 dishes and metformin; n = 17 from 4 dishes. ( c ) Schematic of glycolysis and effects of inhibitors. ( d ) Effects of AR-C155858 (AR-C, 10 μM) and Syrosingopine (Syro, 50 μM) on Perceval fluorescence in Control (red) and Metformin (blue) treated cells. 10 mM glucose (Gluc) was present throughout, and 2-DG (50 mM) added as indicated. Error bars are mean ± SEM. **P < 0.01, ***P < 0.001, two-way ANOVA and Bonferroni post-hoc test. Control: n = 22 cells from 6 dishes; metformin: n = 16 cells from 5 dishes. Error bars are mean ± SEM. **P < 0.01, ***P < 0.001, two-way ANOVA and Bonferroni post-hoc test. ( e ) As ( d ), for oxamate (50 mM). Control: n = 23 cells from 6 dishes; metformin: n = 25 cells from 6 dishes. ( f ) Supernatant lactate levels in control (red) and metformin (blue) pre-treated cultures in glucose (10 mM, 10G), with oxamate (50 mM) or AR-C, (10 μM) and Syro (50 μM). Error bars are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, vs control. ††† P < 0.001 vs pre-treated conditions in 10G. Two-way ANOVA and Bonferroni post-hoc test. n = 2–3 wells from 4 independent experiments except AR–C/Syro (3 independent experiments). ( g ) Effects of oxamate on Peredox fluorescence in control (red) and metformin (blue) treated cells. Glucose (Gluc, 10 mM) and oxamate (50 mM) were applied as indicated. Error bars are median ± 95% confidence intervals. *P < 0.05, Mann–Whitney test. Control: n = 15 cells from 4 dishes; Metformin: n = 18 cells from 4 dishes.

Article Snippet: Transfected cells expressing Perceval HR (21737, Addgene) were imaged at 490 ± 2 nm and 450 nm excitation sequentially, and images were acquired every 10 s. Images were background subtracted using MetaFluor software.

Techniques: Control, Protein Concentration, BIA-KA, Fluorescence, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Inhibition of mitochondrial function by metformin increases glucose uptake, glycolysis and GDF-15 release from intestinal cells

doi: 10.1038/s41598-021-81349-7

Figure Lengend Snippet: Metformin causes mitochondrial dysfunction in intestinal cells. ( a ) Left: Changes in oxygen consumption rate (OCR) during the mitochondrial stress test. Cells were pre-treated with control (ENR media) ± metformin (1 mM) for 24 h, then incubated with glucose (10 mM) in XF basal media for 1 h prior to measurements. Dotted lines indicate compound addition. Oligomycin-A (OliA, 1 μM), FCCP (1 μM), Rotenone/Antimycin A (Rot/AA, 1 μM each). Results were normalised to the protein concentration measured by BCA assay. Right: Effect of metformin on measured mitochondrial respiration parameters. n = 5 wells from 3 independent experiments. ***P < 0.001. Two-way ANOVA and Bonferroni post-hoc test. ( b ) Effects of mitochondrial pyruvate carrier inhibitor UK-5099 compared to DMSO on OCR of control and metformin overnight pre-treated cells as measured by the Seahorse bioanalyser. Results were normalised to the protein concentration measured by BCA assay. Inset: Normalised OCR was calculated as differences after drug application and third time point measured before application ***P < 0.001 compared to DMSO in control cultures. n = 4–5 wells from 4 independent experiments. ( c ) Left: Example traces of Perceval fluorescence ratios from control and metformin treated cells. Glucose (Gluc, 10 mM), Rot/AA (1 μM) and 2-DG (50 mM) were applied as indicated. Right: Effect of 10 mM glucose (Gluc), Rot/AA and 2-DG on the Perceval fluorescence ratios in control (red) and metformin (blue) overnight pre-treated cells (***P < 0.001, Repeated-measures ANOVA and Bonferroni post-hoc test. Control: n = 14 from 5 dishes, metformin: n = 20 cells from 4 dishes). ( d ) Heatmaps showing differentially expressed genes (filtered by p-adjusted value of < 0.05) involved in mitochondrial function based from the MitoCarta 2.0 dataset. Relative expression is normalised to the z-scores for each gene. Each box indicates a separate plate and the corresponding organoid line. Analysis was performed using Bioconductor software packages in RStudio (v.1.2.5019), and the heatmap generated using Pheatmap. ( e ) Confocal images of intestinal cells treated with control or metformin (1 mM) stained with MitoTracker green. Scale bar: 50 µm. Magnified images of individual cells are shown. ( f ) Quantification of the percentage of total cells with fragmented, intermediate or elongated mitochondrial in control or metformin treated cells (***P < 0.001, Chi-square test. Control: n = 1660 cells and metformin: n = 2155 cells, from 3 organoid lines). ( g ) Quantification of mitochondrial morphology as the width/length ratio or roundness by imposition of spots to image regions using the Harmonizer software.

Article Snippet: Transfected cells expressing Perceval HR (21737, Addgene) were imaged at 490 ± 2 nm and 450 nm excitation sequentially, and images were acquired every 10 s. Images were background subtracted using MetaFluor software.

Techniques: Control, Incubation, Protein Concentration, BIA-KA, Fluorescence, Expressing, Software, Generated, Staining