Journal: Oxidative Medicine and Cellular Longevity

Article Title: Impaired Mitochondrial Fusion and Oxidative Phosphorylation Triggered by High Glucose Is Mediated by Tom22 in Endothelial Cells

doi: 10.1155/2019/4508762

Figure Lengend Snippet: Effect of high glucose on the expression of genes regulating mitochondrial dynamics and mitochondrial function. (a) Representative Western blot and quantification analysis of the level of proteins regulating mitochondrial dynamics in each group. The expression of Fis1 and Mff increased significantly upon treatment with high glucose, whereas the expression of Mfn1 decreased significantly. No significant change was seen in the level of Mfn2, Opa1, or Drp1 ( n = 4). (b) Representative Western blot and quantification analysis of the level of proteins that are involved in OXPHOS; the loading control was β -tubulin ( n = 4). (c) Total ATP level in HUVECs exposed to different concentrations of glucose for 48 hr ( n = 4). (d) MMP was analyzed by staining with JC1 and was quantified based on fluorescence intensities. MMP: mitochondrial membrane potential ( n = 3). (e) Apoptotic cells were detected at 48 h by Annexin V/PI staining and flow cytometric analysis ( n = 4) ( ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001 vs. control). Control: normal culture medium; HG15: D-glucose 15 mmol/l; and HG30: D-glucose 30 mmol/l. All data are presented as the means ± SEMs.

Article Snippet: For MMP detection, HUVECs were harvested with 0.25% trypsin-EDTA and resuspended in 0.5 ml culture medium at approximately 2 × 10 6 cells/ml, followed by incubation with 0.5 ml JC1 working solution (Beyotime, China) at 37°C and 5% CO 2 for 20 min. For the positive control condition, 10 μ M CCCP was used to pretreat the cells at 37°C for 20 min.

Techniques: Expressing, Western Blot, Control, Staining, Fluorescence, Membrane

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Impaired Mitochondrial Fusion and Oxidative Phosphorylation Triggered by High Glucose Is Mediated by Tom22 in Endothelial Cells

doi: 10.1155/2019/4508762

Figure Lengend Snippet: Tom22 overexpression attenuated the mitochondrial dysfunction and apoptosis induced by high glucose. HUVECs were transduced with LV-Tom22 vectors that expressed Tom22. The LV-Con vector was used as a vehicle control. (a) Western blot and quantification analysis of the level of proteins regulating mitochondrial dynamics. The expression of Mfn1 decreased significantly, and the expression of both Fis1 and Mff increased significantly. No significant change in the expression of Mfn2, Opa1, or Drp1 was seen ( n = 4). (b) Representative Western blot and quantification analysis of the level of proteins that involve in OXPHOS ( n = 4). (c) Total ATP level in HUVECs ( n = 4). (d) The MMP was analyzed by JC1 staining and quantified based on fluorescence intensities ( n = 3). (e) Apoptotic cells were detected by Annexin V/PI staining ( n = 3). All data are presented as means ± SEMs ( ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001 vs. LV-Con; # P < 0.05 and ## P < 0.01 vs. LV-Con+HG30).

Article Snippet: For MMP detection, HUVECs were harvested with 0.25% trypsin-EDTA and resuspended in 0.5 ml culture medium at approximately 2 × 10 6 cells/ml, followed by incubation with 0.5 ml JC1 working solution (Beyotime, China) at 37°C and 5% CO 2 for 20 min. For the positive control condition, 10 μ M CCCP was used to pretreat the cells at 37°C for 20 min.

Techniques: Over Expression, Transduction, Plasmid Preparation, Control, Western Blot, Expressing, Staining, Fluorescence

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Impaired Mitochondrial Fusion and Oxidative Phosphorylation Triggered by High Glucose Is Mediated by Tom22 in Endothelial Cells

doi: 10.1155/2019/4508762

Figure Lengend Snippet: Downregulation of Mfn1 decreases mitochondrial fusion and induces mitochondrial dysfunction. HUVECs were transfected with Mfn1 siRNA to knock down the expression of Mfn1. NC represents the negative control. (a) The downregulation of Mfn1 was confirmed by real-time PCR ( n = 3). (b) The expression of the Mfn1 protein was detected by Western blotting and quantification analysis ( n = 3). (c) Representative confocal microscopy images of Mfn1 knockdown HUVECs stained with MitoTracker® Deep Red. Scale bar: 10 μ m. (d) Quantification of the form factor and aspect ratio of the mitochondrial networks ( n = 4). (e) Representative images of mitochondria visualized by transmission electron microscopy. Mitochondria shortened in length in the Mfn1 knockdown group. Scale bar: 200 nm. (f) Total ATP level in HUVECs. (g) MMP was analyzed by JC1 staining and quantified based on fluorescence intensities ( n = 3). (h) Apoptotic cells were detected by Annexin V/PI staining ( n = 4). All data are presented as means ± SEMs ( ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001 vs. NC).

Article Snippet: For MMP detection, HUVECs were harvested with 0.25% trypsin-EDTA and resuspended in 0.5 ml culture medium at approximately 2 × 10 6 cells/ml, followed by incubation with 0.5 ml JC1 working solution (Beyotime, China) at 37°C and 5% CO 2 for 20 min. For the positive control condition, 10 μ M CCCP was used to pretreat the cells at 37°C for 20 min.

Techniques: Transfection, Knockdown, Expressing, Negative Control, Real-time Polymerase Chain Reaction, Western Blot, Confocal Microscopy, Staining, Transmission Assay, Electron Microscopy, Fluorescence

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Impaired Mitochondrial Fusion and Oxidative Phosphorylation Triggered by High Glucose Is Mediated by Tom22 in Endothelial Cells

doi: 10.1155/2019/4508762

Figure Lengend Snippet: Overexpression of Mfn1 could protect against pathological mitochondrial dynamics and dysfunction of HUVECs induced by high glucose. HUVECs were transfected with LV-Mfn1 vectors that expressed Mfn1. The LV-Con vector was used as a vehicle control. (a) Western blot analysis was conducted to evaluate the overexpression efficiency of LV-Mfn1 ( n = 3). (b) Representative confocal microscopy images of HUVECs stained with MitoTracker® Deep Red. Scale bar: 10 μ m. (c) Quantification of the form factor and aspect ratio of the mitochondrial networks ( n = 4). (d) Apoptotic cells were detected by Annexin V/PI staining ( n = 3). (e) Total ATP level in HUVECs ( n = 4). (f) The MMP was analyzed by JC1 staining and quantified based on fluorescence intensities ( n = 3). All data are presented as means ± SEMs ( ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001 vs. LV-Con; # P < 0.05, ## P < 0.01, and ### P < 0.001 vs. LV-Con+HG30).

Article Snippet: For MMP detection, HUVECs were harvested with 0.25% trypsin-EDTA and resuspended in 0.5 ml culture medium at approximately 2 × 10 6 cells/ml, followed by incubation with 0.5 ml JC1 working solution (Beyotime, China) at 37°C and 5% CO 2 for 20 min. For the positive control condition, 10 μ M CCCP was used to pretreat the cells at 37°C for 20 min.

Techniques: Over Expression, Transfection, Plasmid Preparation, Control, Western Blot, Confocal Microscopy, Staining, Fluorescence