Journal: Journal of Biological Chemistry

Article Title: Developmental Control of Apoptosis by the Immunophilin Aryl Hydrocarbon Receptor-interacting Protein (AIP) Involves Mitochondrial Import of the Survivin Protein

doi: 10.1074/jbc.m110.210120

Figure Lengend Snippet: FIGURE 6. Tom20 regulation of mitochondrial import of survivin. A, ali- quots of GST, GST-Tom20, or GST-Tom70 recombinant proteins were mixed with Raji cell extracts, and bead-bound material was analyzed by Western blotting. Bottom panel, Coomassie Blue staining of recombinant GST fusion proteins. B, Raji mitochondrial extract was immunoprecipitated with an anti- body to survivin or IgG, and proteins in pellets or supernatants (unbound) were analyzed by Western blotting. C, recombinant wild-type survivin or the survivin 1–141 mutant and GST-Tom20 beads were mixed with increasing concentration of recombinant AIP, and bead-bound proteins were analyzed by Western blotting (top panel). Bottom panel, GST or GST-Tom20 was incu- bated with recombinant AIP in the presence of increasing concentrations of survivin (up to 0.4 M), and bound proteins were analyzed by Western blot- ting. D, independently established clones (#) of HeLa cells stably transfected with control (Ctrl) or Tom70- or Tom20-directed shRNA were analyzed by Western blotting. E, HeLa cells were transfected with control, non-targeting (Ctrl) or Tom20-directed siRNA and analyzed by Western blotting. TCE, total cell extracts. F, clones of HeLa cells with stable knockdown of Tom70 or Tom20 were treated with 0.5 M staurosporine for 24 h and analyzed for cell viability by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide. DataaremeanS.E.ofreplicates(n3).***,p0.0001.Twodifferentclones per conditions were tested with identical results in two independent experiments.

Article Snippet: Antibodies against AIP (Novus Biologicals), survivin (Novus Biologicals), Tom20 (Santa Cruz Biotechnology), Tom70 (Novus Biologicals), COX-IV (Clontech), Smac (Pro-Sci), -actin (Sigma), and Hsp90 (BD Biosciences) were used.

Techniques: Recombinant, Western Blot, Staining, Immunoprecipitation, Mutagenesis, Concentration Assay, Clone Assay, Stable Transfection, Transfection, Control, shRNA, Knockdown

Journal: Mediators of Inflammation

Article Title: Interleukins-27 Aggravates Liver Injury by Impairing the Antimicrobial Response of Macrophages via the Promotion of Mitochondrial Dysfunction in the Context of Sepsis

doi: 10.1155/mi/6608718

Figure Lengend Snippet: IL-27 increased LPS-induced mitophagy in macrophages. (A) The number of mitochondria was detected using MitoTracker ( n = 4). (B) MitoTracker red fluorescence quantification analysis ( n = 4; Control vs. LPS p < 0.0001, LPS vs. LPS + IL-27 p =0.0004). (C) Western blotting was used to detect the mitophagy-related protein expression ( p -Parkin, Parkin, PINK1, and LC3) ( n = 4). (D–G) Relative statistical analysis of p -Parkin/Parkin ( n = 4; Control vs. LPS p < 0.0001, LPS vs. LPS + IL-27 p =0.1192), PINK1 ( n = 4; Control vs. LPS p =0.0001, LPS vs. LPS + IL-27 p =0.0002), LC3 I ( n = 4; Control vs. LPS p =0.0057, LPS vs. LPS + IL-27 p =0.0649) and LC3 II ( n = 4; Control vs. LPS p =0.0038, LPS vs. LPS + IL-27 p < 0.0001) protein expression. (H) TOM20 and LC3 immunofluorescence staining were used to assess mitophagy ( n = 4). (I) Western blotting was used to detect the hepatic p -Parkin/Parkin, PINK1, LC3 I, and LC3 II ( n = 3) protein expression. (J–M) Statistical analysis of relative hepatic p -Parkin/Parkin ( n = 3; WT vs. CLP p =0.0018, CLP vs. IL-27R −/− +CLP p =0.0065), PINK1 ( n = 3; WT vs. CLP p < 0.0001, CLP vs. IL-27R −/− +CLP p =0.0003), LC3 I ( n = 3; WT vs. CLP p < 0.0001, CLP vs. IL-27R −/− +CLP p < 0.0001) and LC3 II ( n = 3; WT vs. CLP p =0.0154, CLP vs. IL-27R −/− +CLP p =0.0077) protein expression. ⁣ ∗ p < 0.05, ⁣ ∗∗ p < 0.01, ⁣ ∗∗∗ p < 0.001, ⁣ ∗∗∗∗ p < 0.0001. CLP, cecal ligation and puncture; IL, interleukin; LPS, lipopolysaccharide; WT, wild-type.

Article Snippet: Following the block with goat serum at room temperature for 45 min, the cells were incubated overnight at 4°C with primary antibodies targeting TOM20 and LC3 and then with FITC- or CY3-conjugated secondary antibodies at room temperature for 30 min (1:200, Affinity Biosciences).

Techniques: Fluorescence, Control, Western Blot, Expressing, Immunofluorescence, Staining, Ligation

Journal: Oxidative Medicine and Cellular Longevity

Article Title: M1 Microglia Induced Neuronal Injury on Ischemic Stroke via Mitochondrial Crosstalk between Microglia and Neurons

doi: 10.1155/2022/4335272

Figure Lengend Snippet: Mitochondrial changes in activated microglia (M1). (a) Schematic diagram of intracellular mitochondrial fusion and fission process in microglia after OGD/R. (b) Western blot assay of mitochondrial fusion protein (Opa1 and Mfn1), TOM20, and cytochrome c in mitochondria of M0 and M1 microglia ( n = 3). Results are displayed in a form of mean ± SD; ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001. (c) Western blotting findings of mitochondrial fission protein including MFF, Fis1, Mid49, and Mid51 in mitochondria of M0 and M1 microglia ( n = 3). Data presented are mean ± SD; ∗∗ P < 0.01 and ∗∗∗ P < 0.001. (d) Mitochondrial function in M1 microglia and M0 microglia was investigated by determining ATP ( n = 3), mitochondria membrane potential ( n = 3), and ROS ( n = 3). The relative ratio of intracellular ATP was determined by calculating the ratio of level of ATP in M0-BV2 and M1-BV2 cells to level of ATP in M0-BV2 cells. Results are displayed in a form of mean ± SD. ∗∗ P < 0.01 and ∗∗∗ P < 0.001. (e) Morphology of intracellular mitochondria in BV2 cells visualized under TEM, scale bar: 1.0 μ m.

Article Snippet: Rabbit antibodies, such as TOM20 (WL0706), BAX (WL01637), BCL-2 (WL01556), cytochrome c (WL02410), caspase 3 (WL04004), iNOS (WL0992a), VDAC (WL02790), IL-2 (WL03259), TNF- α (WL01581), IL-1 β (WLH3903), and β -actin (WL01372), were procured from Wanleibio in Shenyang, China, while rabbit anti-MFF (AF2365), anti-Mid49 (DF12044), anti-Mid51 (DF12019), anti-Opa1 (DF8587), and anti-Mfn1 (DF7543) were obtained from AFFINITY, Co., Ltd.

Techniques: Western Blot, Membrane

Journal: Oxidative Medicine and Cellular Longevity

Article Title: M1 Microglia Induced Neuronal Injury on Ischemic Stroke via Mitochondrial Crosstalk between Microglia and Neurons

doi: 10.1155/2022/4335272

Figure Lengend Snippet: Functional changes of released mitochondria secreted by activated microglia (M1). (a) Morphological characterization of released mitochondria isolated from culture medium of M0 microglia (Mito/M0-BV2) and M1 microglia (Mito/M1-BV2). Scale bar: 1.0 μ m. (b) Levels of proteins associated with mitochondrial fission in Mito/M0-BV2 and Mito/M1-BV2 based on the western blotting ( n = 3). Results are displayed in a form of mean ± SD; ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001. (c) The expression of associated fusion protein, TOM20, and Cyto C in Mito/M0-BV2 and Mito/M1-BV2 ( n = 3). Results are displayed in a form of mean ± SD, ∗ P < 0.05, and ∗∗∗ P < 0.001. (d) After OGD/R for 24 hours, function of Mito/M0-BV2 and Mito/M1-BV2 was detected by checking ATP ( n = 3), mitochondria membrane potential ( n = 3), and ROS ( n = 3). The relative ratio of extracellular ATP in secreted mitochondria from BV2 cells was determined by calculating the ratio of level of ATP in Mito/M0-BV2 and Mito/M1-BV2 to level of ATP in Mito/M0-BV2. Results are displayed in a form of mean ± SD; ∗ P < 0.05 and ∗∗∗ P < 0.001.

Article Snippet: Rabbit antibodies, such as TOM20 (WL0706), BAX (WL01637), BCL-2 (WL01556), cytochrome c (WL02410), caspase 3 (WL04004), iNOS (WL0992a), VDAC (WL02790), IL-2 (WL03259), TNF- α (WL01581), IL-1 β (WLH3903), and β -actin (WL01372), were procured from Wanleibio in Shenyang, China, while rabbit anti-MFF (AF2365), anti-Mid49 (DF12044), anti-Mid51 (DF12019), anti-Opa1 (DF8587), and anti-Mfn1 (DF7543) were obtained from AFFINITY, Co., Ltd.

Techniques: Functional Assay, Isolation, Western Blot, Expressing, Membrane