Journal: Frontiers in Neurology

Article Title: Leptin Maintained Zinc Homeostasis Against Glutamate-Induced Excitotoxicity by Preventing Mitophagy-Mediated Mitochondrial Activation in HT22 Hippocampal Neuronal Cells

doi: 10.3389/fneur.2018.00322

Figure Lengend Snippet: The effect of leptin on mitophagy induced by glutamate in HT22 cells. (A) The fluorescence intensity of Mtphagy Dye and Lyso Dye in mitochondria of HT22 cells; (B) the quantitative analysis of the average fluorescence intensity of mitochondrial Mtphagy Dye in HT22 cells. * P < 0.05, ** P < 0.01. ns, not significant.

Article Snippet: Mitochondrial proteins were extracted using a Mitochondria Protein Extraction Kit (EnoGene, Nanjing, China).

Techniques: Fluorescence

Journal: Frontiers in Neurology

Article Title: Leptin Maintained Zinc Homeostasis Against Glutamate-Induced Excitotoxicity by Preventing Mitophagy-Mediated Mitochondrial Activation in HT22 Hippocampal Neuronal Cells

doi: 10.3389/fneur.2018.00322

Figure Lengend Snippet: The effect of cyclosporinA (CsA) on glutamate-triggered mitochondrial autophagy. (A) The fluorescence intensity of Mtphagy Dye and Lyso Dye in mitochondria of HT22 cells; (B) the quantitative analysis of the average fluorescence intensity of mitochondrial Mtphagy Dye in HT22 cells; (C) Western blot assay of mitochondrial autophagy protein in HT22 cell; (D) the relative expression (integral optical density) of mitochondrial autophagy protein in HT22 cells. * P < 0.05, ** P < 0.01. ns, not significant.

Article Snippet: Mitochondrial proteins were extracted using a Mitochondria Protein Extraction Kit (EnoGene, Nanjing, China).

Techniques: Fluorescence, Western Blot, Expressing

Journal: Microsystems & Nanoengineering

Article Title: Demarcating the membrane damage for the extraction of functional mitochondria

doi: 10.1038/s41378-018-0037-y

Figure Lengend Snippet: a Cells are introduced into the cross-junction of the microchannel. The stress applied on the cell is optimized to disrupt the cell membrane and release subcellular components, while maintaining the integrity of mitochondria. The overview of the microfluidics chip is shown in the inset. b The applied mean stress, modulated by controlling the volumetric flow rate for a given channel geometry, has been optimized by the maximal protein yield (an indication of quantity of the extracted subcellular contents) and the maximal mitotracker positive events (a hallmark of functional mitochondria). Results were obtained by shredding HEK293 cells (10 6 cells/mL) by a range of shear stress and plotted as mean ± SD ( n = 3 independent experiments). A finite element simulation model was established by COMSOL Multiphysics® to illustrate the fluidic flow at the cross-junction. Give a volumetric flow rate at 60 μl/min, c illustrates the velocity profile and the stagnation point at the centre (where the flow velocity is zero), and d illustrates the stress distribution and the extensional flow fields around the stagnation point, which contributes significantly to the cell deformation and disruption

Article Snippet: The number of strokes for Dounce Homogenizer has been optimized in terms of the total protein yield and percentage of mitochondrial membrane potential prior to the comparison (Supplementary Figure ), whereas the Qiagen Kit mitochondria extraction procedures were conducted following the manufacture’s protocols.

Techniques: Membrane, Functional Assay, Shear, Disruption

Journal: Microsystems & Nanoengineering

Article Title: Demarcating the membrane damage for the extraction of functional mitochondria

doi: 10.1038/s41378-018-0037-y

Figure Lengend Snippet: Concentrations of functional mitochondria (number per unit volume) in the extracted sample were measured by mitotracker staining, extracted by the three approaches. a HEK293 cells, and b C2C12 cells. Results were plotted as mean ± SD ( n = 3 independent experiments, * P < 0.05, ** P < 0.01, *** P < 0.001)

Article Snippet: The number of strokes for Dounce Homogenizer has been optimized in terms of the total protein yield and percentage of mitochondrial membrane potential prior to the comparison (Supplementary Figure ), whereas the Qiagen Kit mitochondria extraction procedures were conducted following the manufacture’s protocols.

Techniques: Functional Assay, Staining

Journal: Microsystems & Nanoengineering

Article Title: Demarcating the membrane damage for the extraction of functional mitochondria

doi: 10.1038/s41378-018-0037-y

Figure Lengend Snippet: a Total protein yield and b concentrations of functional mitochondria obtained from the three extraction methods. Results were plotted as mean ± SD ( n = 3 independent experiments, * P < 0.05, ** P < 0.01)

Article Snippet: The number of strokes for Dounce Homogenizer has been optimized in terms of the total protein yield and percentage of mitochondrial membrane potential prior to the comparison (Supplementary Figure ), whereas the Qiagen Kit mitochondria extraction procedures were conducted following the manufacture’s protocols.

Techniques: Functional Assay, Extraction

Journal: Microsystems & Nanoengineering

Article Title: Demarcating the membrane damage for the extraction of functional mitochondria

doi: 10.1038/s41378-018-0037-y

Figure Lengend Snippet: a Cell disruption efficiency, b total protein yield, and c percentage of functional mitochondria measured when the isotonic buffer and hypotonic buffer were used for mitochondrial extraction using the microscale cell shredder and the Dounce Homogenizer. Experiments were conducted by disrupting HEK293 cells of 10 6 cells/mL and results were plotted as mean ± SD ( n = 3 independent experiments)

Article Snippet: The number of strokes for Dounce Homogenizer has been optimized in terms of the total protein yield and percentage of mitochondrial membrane potential prior to the comparison (Supplementary Figure ), whereas the Qiagen Kit mitochondria extraction procedures were conducted following the manufacture’s protocols.

Techniques: Disruption, Functional Assay, Extraction

Journal: Acta Pharmaceutica Sinica. B

Article Title: A novel PGAM5 inhibitor LFHP-1c protects blood–brain barrier integrity in ischemic stroke

doi: 10.1016/j.apsb.2021.01.008

Figure Lengend Snippet: Target verification of LFHP-1c in endothelial cells. (A) Schematic illustration of target protein capture of LFHP-1c in endothelial cells based on SPR. LFHP-1c was printed on the 3D photo-crosslinking chip via the chip array printer through C–H covalent bond connection, and then the lysates of endothelial cells flowed through the surface of the chip. Finally, the proteins were dissociated from the chip and conducted LC–MS/MS analysis and compared with UniProt database. (B) LFHP-1c binds with ΔN21-PGAM5 in kinetic level determined by SPR, and the K D value was about 0.961 μmol/L. (C) LFHP-1c concentration-dependently inhibited the dephosphorylation activity of ΔN21-PGAM5 at molecular level, n = 6 per group. (D) LFHP-1c inhibited the dephosphorylation activity of PGAM5 in isolated mitochondria from mouse brain-derived Endothelial cells.3 (bEnd.3) in a concentration-dependent manner, n = 3 per group. (E) Schematic illustration of target identification in rBMECs lysates. Photoaffinity probe HP-62 binds with proteome in rBMECs through photoaffinity labeling, and then clicks with biotin-PEG3-N3 based on copper-catalyzed azide–alkyne cycloaddition (CuAAC), subsequently enriched by Streptavidin Mag Sepharose™ beads, and finally separated by SDS-PAGE followed by immunoblotting. (F) Evaluation of HP-62 effect on the dephosphorylation activity of PGAM5 compared to the parent compound LFHP-1c at molecular level, and the results reveal that HP-62 retained the dephosphorylation activity of PGAM5, n = 6 per group. (G) Pull-down/Western blotting for target validation of PGAM5 with the photoaffinity probe HP-62, and a representative blot shown here. Results are expressed as mean ± SEM. ∗ P < 0.05, ∗ ∗ ∗ P < 0.001 versus Control (Ctrl) group.

Article Snippet: The cytoplasmic and mitochondria extraction kit, the cytoplasmic and nuclear protein extraction kit, LDH assay kit, bovine serum albumin (BSA), PBS, HBSS, HEPES, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) apoptosis assay kit, protein A/G agarose and Hoechst 33342 were purchased from Beyotime Biotechnology (Shanghai, China).

Techniques: Liquid Chromatography with Mass Spectroscopy, Concentration Assay, De-Phosphorylation Assay, Activity Assay, Isolation, Derivative Assay, Drug discovery, Labeling, SDS Page, Western Blot, Biomarker Discovery, Control