Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: β-Arrestin2 is a critical component of the GPCR–eNOS signalosome

doi: 10.1073/pnas.1922608117

Figure Lengend Snippet: Superresolution imaging of β-Arr2/eNOS in normal and injured SECs. SECs were isolated form normal (A) and injured (BDL, B) rat livers. β-Arr2 and eNOS were immmunolabeled with mouse monoclonal anti–β-Arr2 and rabbit polyclonal anti-eNOS antibody. β-Arr2 (Alexa Fluor 647, green) and eNOS (Alexa Fluor 568, magenta) were visualized in horizontal cross-section (X-Y), and in cross-section (X-Z) with an SR-200 inverted microscope (Vutara, Inc.) (60×/1.2 NA Olympus water immersion objective). Samples were imaged based on the SML biplane FPALM technology as described. Representative white field (Left) with the portion of the cell that is imaged at high resolution; X-Y section with eNOS labeled in magenta and β-Arr2 in green; and X-Z section images with β-Arr2 and eNOS colocalization appearing white from normal SECs (Upper) and injured SECs (Lower) are shown. (Scale bars, 1 μm.)

Article Snippet: Samples were imaged based on the single molecule localization (SML) biplane fluorescence photoactivation localization microscopy (FPALM) technology as described ( 27 , 28 ) and data were analyzed using Vutara SRX software (version 3.16).

Techniques: Imaging, Isolation, Inverted Microscopy, Labeling

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: β-Arrestin2 is a critical component of the GPCR–eNOS signalosome

doi: 10.1073/pnas.1922608117

Figure Lengend Snippet: ET-1 activation of β-Arr2 and eNOS. (A) SECs from normal (Left two panels) and BDL injured (Right two panels) rat livers were cultured for 24 h and exposed to ET-1 (20 nM) for 30 min. Cells were fixed and labeled with antibody to β-Arr2 (Upper red) and nuclei were labeled with DAPI (Lower, blue). Representative images (of more than 10 others) of single and merged channels are shown. (Scale bars, 10 μm.) (B) SECs isolated from normal rat livers (Left two panels) and BDL injured (Right two panels) rat livers were cultured for 24 h and exposed to ET-1 as in A. β-Arr2 and eNOS were immmunolabeled with anti–β-Arr2 and anti-eNOS antibodies. β-Arr2 (Alexa Fluor 647, green) and eNOS (Alexa Fluor 568, magenta) were visualized in horizontal cross-section (X-Y), in cross-section (X-Z) with an SR-200 inverted microscope (Vutara, Inc.) (60×/1.2 NA Olympus water immersion objective). Samples were imaged based on the SML biplane FPALM technology as described. Representative X-Y section (Upper) and X-Z (Bottom) section images from control (Left) and exposed to ET-1 (Right) are shown. (Scale bar, 1 μm.) (C) β-Arr2 and eNOS colocalization in normal and injured SECs as in B was quantified as in Materials and Methods; the changes in normal and injured SECs are presented graphically (1 for complete and 0 for no colocalization [n = 3/group]). (D) SECs isolated from β-Arr2 WT and KO mice were exposed to ET-1 (20 nM) for 30 min. Cell lysates were subjected to immunoblotting with the indicated antibodies. Specific bands corresponding to P-eNOS were quantified and are presented as the ratio of P-eNOS to eNOS, shown on the Right (n = 3/group). (E) eNOS enzymatic activity was measured in cell lysates treated as in D and NOS activity was normalized to that of control cells from β-Arr2 WT mice without ET-1 exposure and is presented graphically (the activity in SECs from β-Arr2 WT mice without ET-1 exposure was set at 100; n = 3/group). Statistical significance for C–E was evaluated by ordinary one-way ANOVA. Data are mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.005 for differences between indicated groups; n.s., no significant difference.

Article Snippet: Samples were imaged based on the single molecule localization (SML) biplane fluorescence photoactivation localization microscopy (FPALM) technology as described ( 27 , 28 ) and data were analyzed using Vutara SRX software (version 3.16).

Techniques: Activation Assay, Cell Culture, Labeling, Isolation, Inverted Microscopy, Control, Western Blot, Activity Assay

Journal: Frontiers in Pharmacology

Article Title: Effect of Dl-3-n-butylphthalide on mitochondrial Cox7c in models of cerebral ischemia/reperfusion injury

doi: 10.3389/fphar.2023.1084564

Figure Lengend Snippet: Effects of NBP on damage to neurons and the blood brain barrier after ischemia/reperfusion in mice. (A) The mNSS was used to assess neurological deficits. (B) TTC staining showed the infarcted area. (C) Cerebral infarction volume was calculated and quantified using ImageJ. (D) Blood brain barrier permeability was determined based on Evans Blue leakage. (E) Inverted fluorescence microscopy showing TUNEL-stained (green fluorescence) apoptotic neural cells. (F) Fluorescence microscopy shows the expression of the hypoxyprobe (green fluorescence) in the brain tissue of mice to assess the degree of hypoxia. (G–I) Effects of NBP on the protein expression of ZO-1 and occludin in ischemia/reperfusion brain tissue and results of semi-quantitative analysis of protein grey scale. (A–C) n = 6 mice per group. (D–F) n = 5 mice per group. (G–I) the data are representative of three independent experiments. All data are expressed as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001. Scale bars, (D) 100μm, (F) 500 μm.

Article Snippet: The detection of cell apoptosis and mitochondrial localization were performed according to the instructions of the One Step TUNEL Apoptosis Assay Kit (Beyotime, China) and Mito-Tracker Red CMXRos (Beyotime, China).

Techniques: Staining, Permeability, Fluorescence, Microscopy, TUNEL Assay, Expressing

Journal: Frontiers in Pharmacology

Article Title: Effect of Dl-3-n-butylphthalide on mitochondrial Cox7c in models of cerebral ischemia/reperfusion injury

doi: 10.3389/fphar.2023.1084564

Figure Lengend Snippet: Effects of oeCox7c on ischemia/reperfusion models in vivo . (A) Fluorescence microscopy showing Cox7c (red fluorescence) expression in oeCox7c mouse brain and co-localisation with GFP (green fluorescence) (B,C) Effects of oeCox7c on Cox7c expression in MCAO/R mouse brain, and results of semi-quantitative analysis of protein grey scale. (D) The mNSS was used to assess neurological deficits of control and oeCox7c MCAO/R mice. (E) TTC staining shows the infarcted area of control and oeCox7c MCAO/R mice. (F) Cerebral infarction volume was calculated and quantified using ImageJ. (G) Blood brain barrier permeability was determined based on Evans Blue leakage of control and oeCox7c MCAO/R mice. (H) Inverted fluorescence microscopy showing TUNEL-stained (green fluorescence) apoptotic neural cells. The data are representative of three independent experiments. All data are expressed as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001. Scale bars, (A,H) :100 μm.

Article Snippet: The detection of cell apoptosis and mitochondrial localization were performed according to the instructions of the One Step TUNEL Apoptosis Assay Kit (Beyotime, China) and Mito-Tracker Red CMXRos (Beyotime, China).

Techniques: In Vivo, Fluorescence, Microscopy, Expressing, Control, Staining, Permeability, TUNEL Assay

Journal: Frontiers in Pharmacology

Article Title: Effect of Dl-3-n-butylphthalide on mitochondrial Cox7c in models of cerebral ischemia/reperfusion injury

doi: 10.3389/fphar.2023.1084564

Figure Lengend Snippet: Model of the mechanism by which NBP inhibits mitochondrial dysfunction in ischemia/reperfusion injured vascular endothelial cells. Ischemia/reperfusion induces mitochondrial dysfunction in vascular endothelial cells, while NBP can directly target Cox7c on mitochondria, inhibits anaerobic glycolysis, increases the production of ATP by upregulated the expression of Cox7c, promotes angiogenesis by upregulated the HIF-1α/VEGF pathway, increases the expression of TJs (ZO-1, occludin) to protect blood brain barrier. In addition, NBP inhibits the release of ROS, and then protects the mitochondrial membrane potential, inhibits oxidative stress response, suppresses the apoptosis pathway.

Article Snippet: The detection of cell apoptosis and mitochondrial localization were performed according to the instructions of the One Step TUNEL Apoptosis Assay Kit (Beyotime, China) and Mito-Tracker Red CMXRos (Beyotime, China).

Techniques: Expressing, Membrane