Journal: The American journal of pathology

Article Title: Hyperglycemia Promotes Mitophagy and Thereby Mitigates Hyperglycemia-Induced Damage.

doi: 10.1016/j.ajpath.2022.08.004

Figure Lengend Snippet: Figure 4 Mitophagy is elevated in adapted cells. A: Representative confocal images showing the overlay of red and green fluorescence in MtKeima- expressing human retinal endothelial cells (HRECs) cultured in normal glucose (NG) or high glucose (HG). NG cells treated with FCCP (10 mmol/L), a mito- chondrial membrane protonophore, were used as a positive control. The red signals (white arrows) point to mitochondria within an acidic compartment, such as the lysosome. B: The extent of mitophagy in MtKeima-expressing HRECs subjected to the indicated experimental conditions was quantified. The data presented are from at least three independent experiments, which were normalized to the level of mitophagy in NG-HRECs. C: PTEN induced kinase 1 (PINK1), transcription factor A, mitochondrial (TFAM), and translocase of outer mitochondrial membrane 20 (TOMM20) mRNA levels in HRECs cultured in NG or HG. D: Cytochrome C oxidase subunit 4I1 (COX4) and TOMM20 protein levels in HRECs treated with NG or HG. The signal was quantified using ImageJ software (version 2.0.0-rc-69/1.52v; build 269a0ad53f); COX4 and TOMM20 were normalized to b-actin. Each experiment included triplicate samples. Data indicate the average SD of triplicates of a single representative experiment (C) or the average SD of three independent experiments (D). Statistical significance was calculated by two-tailed t-test with unpaired two-sample equal variance (B). n Z 3 (C). *P < 0.05. Scale bar Z 20 mm (A).

Article Snippet: To measure the total level of mitochondria, antibodies translocase of outer mitochondrial membrane 20 (TOM20; D8T4N rabbit mAb; number 42406; 1:1000 dilution; Cell Signaling) and cytochrome C oxidase subunit 4I1 (COXIV; 3E11; rabbit mAb; number 4850; 1:1000 dilution; Cell Signaling) were used.

Techniques: Expressing, Cell Culture, Membrane, Positive Control, Software, Two Tailed Test

Journal: Microbiome

Article Title: The microbiota-gut-brain axis participates in chronic cerebral hypoperfusion by disrupting the metabolism of short-chain fatty acids

doi: 10.1186/s40168-022-01255-6

Figure Lengend Snippet: FMT ameliorates BCCAO-induced hippocampal neuronal apoptosis and LPS-stimulated neuroinflammation. A Representative immunofluorescence images of hippocampal neurons in CA1, CA2, CA3, and DG zones. Scale bar, 50 μm. B Bar plots of the mean fluorescence intensity in the different groups ( n = 6 S+FMT(B) and S+FMT(S), n = 8 B+FMT(B) and B+FMT(S)). C mRNA levels of inflammatory cytokines ( TNF-α , IL-1β , IL-6 , IL-4 , and IL-10 ) in the hippocampus ( n = 5/group). D – G Western blot analysis for protein levels of caspase 3, cleaved caspase 3, Bcl2, Bax, Bcl-XL, Erk, and pErk and their quantification (band intensity normalized to β-actin or COXIV) in the hippocampus ( n = 6/group). H – K Representative immunofluorescence images showing Iba-1+ cells in the cortex ( H ) and hippocampus ( J ), and the quantification analysis ( I , K ) ( n = 5/group). Scale bar, 50 μm. L mRNA levels of inflammatory cytokines ( TNF-α , IL-1β , IL-6 , IL-4 , and IL-10 ) in the cortex ( n = 5/group). M , N Protein levels of CD16 and NF-κB and their quantification (band intensity normalized to β-actin) in the cortex ( n = 5/group). S+FMT(B)+LPS, sham rats treated with LPS after receiving BCCAO-rat-derived fecal microbiota transplantation. S+FMT(S)+LPS, sham rats treated with LPS after receiving sham-rat-derived fecal microbiota transplantation. The data represent the mean ± SEM. p < 0.05 was set as the threshold for significance. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to the S+FMT(B) group. & p < 0.05, && p < 0.01, &&& p < 0.001 compared to the S+FMT(S) group. # p < 0.05, ## p < 0.01 compared to the B+FMT(B) group. + p < 0.05, ++ p < 0.01 compared to the S+FMT(B)+LPS group

Article Snippet: The primary antibodies used included rabbit polyclonal anti-caspase 3 (1:1,000, 19677-1-AP; Proteintech, USA), rabbit polyclonal anti-Bcl 2 (1:1,000, 12789-1-AP; Proteintech, USA), rabbit polyclonal anti-Bax (1:1,000, 50599-2-Ig; Proteintech, USA), rabbit polyclonal anti-Bcl-XL (1:1,000, 26967-1-AP; Proteintech, USA), rabbit monoclonal anti-cytochrome c oxidase subunit IV isoform (COXIV) (1:5,000, 66110-1-Ig; Proteintech, USA), rabbit monoclonal anti-phospho-Erk1/2 (1:1,000, #4370; Cell Signaling Technology, USA), rabbit monoclonal anti-Erk1/2 (1:1,000, #4695; Cell Signaling Technology, USA), rabbit monoclonal anti-NF-κB p65 (1:1,000, #8242; Cell Signaling Technology, USA), rabbit monoclonal anti-CD16 (1:1,000, ab211151; Abcam, England), and mouse monoclonal anti-β-Actin (1:5,000, #3700; Cell Signaling Technology, USA).

Techniques: Immunofluorescence, Fluorescence, Western Blot, Derivative Assay, Transplantation Assay

Journal: Microbiome

Article Title: The microbiota-gut-brain axis participates in chronic cerebral hypoperfusion by disrupting the metabolism of short-chain fatty acids

doi: 10.1186/s40168-022-01255-6

Figure Lengend Snippet: SCFA supplementation mitigates neuroinflammation and promotes neuronal survival in the hippocampus post BCCAO. A , B Representative immunofluorescence images showing Iba-1+ cells in the hippocampus at day 14 post BCCAO ( A ) and the quantification analysis ( B ) ( n = 6/group). Scale bar, 50 μm. C , D mRNA levels of inflammatory cytokines ( IL-4 , IL-10, TNF-α , IL-1β , and IL-6 ) in the hippocampus at day 14 post BCCAO ( n = 5/group). E Protein levels of CD16 and NF-κB and their quantification in the hippocampus at day 14 post BCCAO (band intensity normalized to β-actin) ( n = 6/group). F , G Representative immunofluorescence images ( F ) and mean fluorescence intensity ( G ) of hippocampal neurons in CA1, CA2, CA3, and DG in the different groups. ( n = 6/group). Scale bar, 50 μm. H – K Western blot analysis for protein levels of caspase 3, cleaved caspase 3, Bcl2, Bax, Bcl-XL, Erk, and pErk and their quantification (band intensity normalized to β-actin or COXIV) in the hippocampus ( n = 6/group). The data represent the mean ± SEM. p < 0.05 was set as the threshold for significance. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to S+NaCl group. && p < 0.01 compared to the S+SCFA group. + p < 0.05 compared to the B+NaCl 14d group. # p < 0.05, ## p < 0.01 compared to the B+NaCl 37d group

Article Snippet: The primary antibodies used included rabbit polyclonal anti-caspase 3 (1:1,000, 19677-1-AP; Proteintech, USA), rabbit polyclonal anti-Bcl 2 (1:1,000, 12789-1-AP; Proteintech, USA), rabbit polyclonal anti-Bax (1:1,000, 50599-2-Ig; Proteintech, USA), rabbit polyclonal anti-Bcl-XL (1:1,000, 26967-1-AP; Proteintech, USA), rabbit monoclonal anti-cytochrome c oxidase subunit IV isoform (COXIV) (1:5,000, 66110-1-Ig; Proteintech, USA), rabbit monoclonal anti-phospho-Erk1/2 (1:1,000, #4370; Cell Signaling Technology, USA), rabbit monoclonal anti-Erk1/2 (1:1,000, #4695; Cell Signaling Technology, USA), rabbit monoclonal anti-NF-κB p65 (1:1,000, #8242; Cell Signaling Technology, USA), rabbit monoclonal anti-CD16 (1:1,000, ab211151; Abcam, England), and mouse monoclonal anti-β-Actin (1:5,000, #3700; Cell Signaling Technology, USA).

Techniques: Immunofluorescence, Fluorescence, Western Blot

Journal: Journal of Neuroinflammation

Article Title: Exercise-induced irisin ameliorates cognitive impairment following chronic cerebral hypoperfusion by suppressing neuroinflammation and hippocampal neuronal apoptosis

doi: 10.1186/s12974-025-03493-5

Figure Lengend Snippet: Effects of increasing endogenous irisin by FAE on hippocampal neuronal apoptosis and white matter injury triggered by CCH. A , B Representative immunofluorescence images of NeuN + hippocampal neurons in CA1, CA2, and CA3 zones, and their quantification per field ( n = 6/group). Scale bar, 50 μm. C-E Western blot analysis of apoptosis executor protein levels, including caspase 3 and cleaved caspase-3 expression in the hippocampus after CCH, with quantification (band intensity normalized to β-actin) ( n = 6/group). F-H Western blot analysis of apoptosis associated protein levels, including Bcl2, Bax, Bcl-XL expression in the hippocampus after CCH, with quantification (band intensity normalized to COXIV) ( n = 6/group). I , J Representative immunofluorescence images of MBP + cortex, corpus callosum, and striatum zones, and their quantification of mean fluorescence intensity ( n = 5/group). Scale bar, 100 μm. One-way ANOVA, Tukey post hoc test ( B–J ). The data represent the mean ± SD, p < 0.05 was set as the threshold for significance. * p < 0.05, ** p < 0.01, *** p < 0.001, ns, no significance, as indicated

Article Snippet: The primary antibodies used in this study included rabbit monoclonal anti-FNDC5 (1:1000, ab174833; Abcam, England), rabbit monoclonal anti-integrin αV (1:1000, ab179475; Abcam, England), rabbit monoclonal anti-integrin β5 (1:1000, #3629; Cell Signaling Technology, USA), rabbit polyclonal anti-caspase 3 (1:1,000, 19677-1-AP; Proteintech, China), rabbit monoclonal anti-Fc RII/III receptor (CD16) (1:1,000, ab211151; abcam, England), rabbit monoclonal anti-CD21 (C3d) (1:1,000, SC0681; HUABIO, China), rabbit polyclonal anti-Bcl 2 (1:1,000, 12789-1-AP; Proteintech, China), rabbit polyclonal anti-Bax (1:1,000, 50599-2-Ig; Proteintech, China), rabbit polyclonal anti-Bcl-XL (1:1,000, 26967-1-AP; Proteintech, China), rabbit monoclonal anti-cytochrome c oxidase subunit IV isoform (COXIV) (1:5,000, 66110-1-Ig; Proteintech, China), rabbit polyclonal anti-nitric oxide synthase 2, inducible (iNOS) (1:500, BA0362; Boster, China), rabbit polyclonal anti-CD86 (1:500, 13395-1-AP; Proteintech, China), rabbit polyclonal anti-TNF-alpha (1:500, 17590-1-AP; Proteintech, China), rabbit polyclonal anti-Sequestosome-1 (P62) (1:1000, 18420-1-AP; Proteintech, China), rabbit polyclonal anti-Beclin 1/BECN1 (1:500, PB9076; Boster, China), rabbit polyclonal anti-MAP1LC3B (1:1000, L7543; Sigma-Aldrich, USA), rabbit monoclonal anti-phospho-mTOR (pmTOR) (phospho S2448) (1:1,000, ab109268; abcam, England), rabbit polyclonal anti-mTOR (1:1000, 28273-1-AP; Proteintech, China), rabbit monoclonal anti-phospho-AMP-activated protein kinase α (pAMPKα) (1:1000, #2535; Cell Signaling Technology, USA), rabbit monoclonal anti-AMPKα (1:1000, #5831; Cell Signaling Technology, USA), mouse monoclonal anti-β-actin (1:5000, 66009-1-Ig; Proteintech, China).

Techniques: Immunofluorescence, Western Blot, Expressing, Fluorescence