Journal: Nature Communications

Article Title: GATD3A-deficiency-induced mitochondrial dysfunction facilitates senescence of fibroblast-like synoviocytes and osteoarthritis progression

doi: 10.1038/s41467-024-55335-2

Figure Lengend Snippet: a The heatmap of RNA-Seq analysis for FLSs separated from normal and OA patients. b The biological processes of differentially expressed mRNAs were analysed by GO enrichment analysis. c GSEA of ‘OXPHOS’ gene sets in Nor-FLSs and OA-FLSs. (d) Representative images of Mitotracker staining to observe the mitochondrial network of Nor-FLSs ( n = 12) and OA-FLSs ( n = 11), and the statistical analysis of mean branch length. Scale bar: 40 µm. The white dashed box represents the enlarged image area in the bottom right corner. e Representative images of TEM to observe Nor-FLSs ( n = 5) and OA-FLSs ( n = 12) and the arrows point to the mitochondria. Scale bar: 1 µm. f Representative images of MitoSOX staining to detect mitochondrial superoxide in Nor-FLSs and OA-FLSs. n = 6 independent biological replicates. Scale bar: 100 µm. g Mitochondrial membrane potential detected by JC-1 assay and the statistical analysis of aggregate-to-monomer ratio ( n = 3 Nor-FLSs, n = 7 OA-FLSs). h , i OCR of Nor-FLSs and OA-FLSs, and the analysis of mitochondrial respiration. n = 3 independent biological replicates. j , k PER of Nor-FLSs and OA-FLSs, and the analysis of basal and compensatory glycolysis. n = 4 independent biological replicates. l Quantification of ATP content in Nor-FLSs and OA-FLSs. n = 5 independent biological replicates. All data were presented as the means ± SD. P values were determined by two-tailed unpaired Student’s t -test. P < 0.05 was considered statistically significant. Source data are provided as a Source Data file.

Article Snippet: To obtain the mitochondria of cells, a Mitochondrial Isolation kit (MCE, USA) was used.

Techniques: RNA Sequencing, Staining, Membrane, Two Tailed Test

Journal: Advanced Science

Article Title: Celastrol Ameliorates Neuronal Mitochondrial Dysfunction Induced by Intracerebral Hemorrhage via Targeting cAMP‐Activated Exchange Protein‐1

doi: 10.1002/advs.202307556

Figure Lengend Snippet: Celastrol improved neuronal mitochondrial dysfunction induced by ICH. A) The chemical structure of celastrol labeled with biotin is illustrated. B) Neurons were treated with 50 n m biotin or 50 n m Biotin‐Cel for 6 h, followed by immunofluorescence analysis using an anti‐biotin antibody (green) in neurons and mitotracker staining for mitochondria (red). Nuclei were stained with DAPI (blue). Representative images from triplicate experiments are shown. Arrows indicate the co‐localization of celastrol and mitochondria. Scale bar: 10 µm. C) Transmission electron microscopy of mitochondrial structures in neuronal cells of mice across different experimental groups. Scale bar: 5 µm. The neurons were stimulated with 10 µ m OxyHb for 12 h and subsequently exposed to varying doses of celastrol treatment for 24 h. Afterward, the cells were collected for assessment of mitochondrial function. D) Oxygen consumption rates (OCR) were measured by using the Seahorse XF‐24 Extracellular Flux Analyze, n = 3. E) Extracellular acidification rate (ECAR) was measured by using the Seahorse XF‐24 Extracellular Flux Analyze, n = 3. F) The ATP content was measured using a chemiluminescence assay, n = 6. G) Neurons from various experimental groups were subjected to JC‐1 staining and subsequently visualized under a Nikon fluorescence microscope. Scale bar: 100 µm. H) Neurons from different experimental groups were subjected to JC‐1 staining, and the fluorescence intensity was quantified using a fluorescent microplate reader with excitation/emission wavelengths of 514/529 nm for monomers and 585/590 nm for aggregates, n = 6. I) Cytoplasm and mitochondria were separated to detect the protein levels of Cyto C, which was normalized to 1.0 based on the mean value of each protein in the control group. β‐Tubulin and voltage‐dependent anion‐selective channel protein 1 (VDAC1) served as loading controls. J) Neurons from various experimental groups were lysed, and western blot analysis was performed to determine the protein levels of caspase‐9. The mean value of each protein in the control group was normalized to 1.0, with GAPDH serving as the loading control, n = 6. K) Neurons from various experimental groups were lysed, and western blot analysis was performed to determine the protein levels of caspase‐3. The mean value of each protein in the control group was normalized to 1.0, with β‐Tubulin serving as the loading control, n = 6. All data are presented as mean ± SD. Statistical significance was determined using one‐ or two‐way ANOVA with Tukey's multiple comparisons tests ( * p < 0.05, ** p < 0.001, *** p < 0.0001 vs control group; # p < 0.05, ## p < 0.001, ### p < 0.0001 vs vehicle group).

Article Snippet: Mitochondria were extracted using the Tissue and Cell Mitochondrial Extraction Kit (Beyotime, C3606, C3601) and separated from the cytoplasm through differential centrifugation of fresh brain tissue (processed within 1 h) and neuronal cells according to the manufacturer's instructions.

Techniques: Labeling, Immunofluorescence, Staining, Transmission Assay, Electron Microscopy, Chemiluminescence Immunoassay, Fluorescence, Microscopy, Control, Western Blot

Journal: Advanced Science

Article Title: Celastrol Ameliorates Neuronal Mitochondrial Dysfunction Induced by Intracerebral Hemorrhage via Targeting cAMP‐Activated Exchange Protein‐1

doi: 10.1002/advs.202307556

Figure Lengend Snippet: EPAC‐1 contributes to mitochondrial dysfunction induced by ICH through its interaction with VDAC1. A) Samples of cortex surrounding the hematoma in mice were collected at 3, 6, 12, 24, and 48 h post‐ICH induction. The protein levels of EPAC‐1 were assessed via western blot analysis, and the alterations in expression were quantified. β‐Tubulin served as loading controls, with a sham group being normalized to a value of 1.0 for accurate comparisons ( n = 6). B) EPAC‐1 activation was assessed by measuring the levels of activated Rap1‐GTP using a Rap1 Activation Assay Kit. β‐Tubulin served as loading controls, with each sham group being normalized to a value of 1 for accurate comparisons n = 6. C) Neurons underwent a 12 h stimulation with 10 µ m OxyHb. Subsequently, mitochondria and cytoplasm were isolated, and the protein levels of EPAC‐1 in both compartments were evaluated using western blot analysis. β‐Tubulin and Tom20 served as loading controls, with each control group being normalized to a value of 1 for accurate comparisons, n = 6. D) Protein‐protein interaction (PPI) network was queried from the STRING database ( https://string‐db.org ), followed by functional protein enrichment analysis conducted through Cytoscape and ClueGO. E) Rigid protein‐protein docking (ZDOCK) was conducted to investigate the relationship between EPAC‐1 and VDAC1. The PDB format of the protein structural domain was obtained from the Protein Data Bank (PDB) at http://www.rcsb.org/ . The ZDOCK module was used to identify docking sites and calculate ZDOCK scores. F) Cortex samples surrounding the hematoma in mice were collected from sham and ICH 24 h groups, followed by co‐immunoprecipitation to validate the interaction between EPAC‐1 and VDAC‐1. G,H) Following stimulation with 10 µ m OxyHb for 12 h, neurons were treated with the EPAC‐1 inhibitor ESI09 (10 µ m ) or activator 8CPT (10 µ m ) for 24 h. The levels of activated Rap1‐GTP were detected using a Rap1 Activation Assay Kit (G), while EPAC‐1 protein levels in both compartments were assessed via western blot analysis (H). Co‐immunoprecipitation was used to detect the interaction between EPAC‐1 and VDAC‐1 I), and mitochondrial structures were examined by means of transmission electron microscopy with a scale bar of 5 µm J). The ATP content was quantified L), and mitochondrial membrane potential (MMP) was measured by JC‐1 staining K). All data are presented as mean ± SD. Statistical significance was determined using one‐ or two‐way ANOVA with Tukey's multiple comparisons tests ( n = 6, *** p < 0.0001 vs control/ sham group; ### p < 0.0001 vs Vehicle group).

Article Snippet: Mitochondria were extracted using the Tissue and Cell Mitochondrial Extraction Kit (Beyotime, C3606, C3601) and separated from the cytoplasm through differential centrifugation of fresh brain tissue (processed within 1 h) and neuronal cells according to the manufacturer's instructions.

Techniques: Western Blot, Expressing, Activation Assay, Isolation, Control, Functional Assay, Protein Enrichment, Immunoprecipitation, Transmission Assay, Electron Microscopy, Membrane, Staining

Journal: Advanced Science

Article Title: Celastrol Ameliorates Neuronal Mitochondrial Dysfunction Induced by Intracerebral Hemorrhage via Targeting cAMP‐Activated Exchange Protein‐1

doi: 10.1002/advs.202307556

Figure Lengend Snippet: Celastrol inhibits the elevation of EPAC‐1 activity and MPTP opening induced by ICH. A) Samples of cortex surrounding the hematoma in mice were collected 48 h after ICH induction and treatment with 1, 2, and 4 mg k −1 g celastrol. EPAC‐1 protein levels were detected by western blot. β‐Tubulin served as loading controls, with a sham group being normalized to a value of 1 for accurate comparisons, n = 6. B) After being stimulated with OxyHb, the neurons were treated with the EPAC‐1 inhibitor ESI09 (10 µ m ) or celastrol at concentrations of 25 and 50 n m . The activation levels of Rap1‐GTP were measured using a Rap1 Activation Assay Kit. β‐Tubulin served as loading controls, with each sham group being normalized to a value of 1 for accurate comparisons, n = 6. C) Following stimulation with OxyHb, the neurons were treated with 50 n m celastrol. Immunofluorescence analysis was performed using an anti‐EPAC‐1 antibody (green) to label neurons and mitotracker staining for mitochondria (red). Nuclei were counterstained with DAPI (blue). Representative images from triplicate experiments are presented, showing co‐localization of celastrol and mitochondria in the proximal neurite, as indicated by arrows. Scale bar: 10 µm. D) The protein levels of EPAC‐1 in the mitochondria and cytoplasm were evaluated by western blot analysis across different groups. The mean value of each protein in the control group was normalized to 1.0, with β‐tubulin and Tom 20 serving as the loading control, n = 6. E) Co‐immunoprecipitation was used to detect the interaction between EPAC‐1 and VDAC‐1. F) Mitochondria were pretreated with celastrol or ESI09 for 30 min, followed by exposure to Ca 2+ for another 10 min. Mitochondria swelling traces were recorded based on the absorbance at 540 nm. The mitochondrial swelling was quantified, n = 6. The neurons were stimulated with 10 µ m OxyHb for 12 h and subsequently exposed to varying doses of celastrol treatment for 24 h. Afterward, the cells were collected for assessment of mitochondrial function. G) Mitochondrial structures in neurons, with or without celastrol treatment following OxyHb stimulation, were observed using transmission electron microscopy. The scale bar was set at 5 µm. H) Neurons from various experimental groups were subjected to MitoSOX staining and subsequently visualized under a Nikon fluorescence microscope. I) Neurons from various experimental groups were subjected to MitoSOX staining, and the fluorescence intensity was quantified using a fluorescent microplate reader with excitation/emission wavelengths of 396/610 nm, n = 6. J) The Fluo3‐AM staining was used to detect calcium in the cytoplasm of neurons, which was subsequently visualized using a Nikon fluorescence microscope. K) The fluorescence intensity of Fluo3‐AM was measured using a fluorescent microplate reader with excitation and emission wavelengths set at 506 and 526 nm, respectively, n = 6. All data are presented as mean ± SD. Statistical significance was determined using one‐or two‐way ANOVA with Tukey's multiple comparisons tests ( * p < 0.05, ** p < 0.001, *** p < 0.0001 vs control group; # p < 0.05, ## p < 0.001, ### p < 0.0001 vs vehicle group).

Article Snippet: Mitochondria were extracted using the Tissue and Cell Mitochondrial Extraction Kit (Beyotime, C3606, C3601) and separated from the cytoplasm through differential centrifugation of fresh brain tissue (processed within 1 h) and neuronal cells according to the manufacturer's instructions.

Techniques: Activity Assay, Western Blot, Activation Assay, Immunofluorescence, Staining, Control, Immunoprecipitation, Transmission Assay, Electron Microscopy, Fluorescence, Microscopy

Journal: Advanced Science

Article Title: Celastrol Ameliorates Neuronal Mitochondrial Dysfunction Induced by Intracerebral Hemorrhage via Targeting cAMP‐Activated Exchange Protein‐1

doi: 10.1002/advs.202307556

Figure Lengend Snippet: Graphic illustration of neuroprotective effects and mechanisms of celastrol. Following ICH, EPAC‐1 is activated within neurons and translocated to the outer membrane of mitochondria. Here, it can form a complex with VDAC1, promoting MPTP opening and subsequent collapse of mitochondrial membrane potential. This results in Ca 2+ release, which induces neuronal apoptosis via cytochrome C (Cyto C). As a natural compound, celastrol can directly localize in mitochondria and interact with EPAC‐1 to modulate its activation, thereby impeding the binding of EPAC‐1 to VADC1. This ameliorates mitochondrial impairment in neuronal cells and exerts neuroprotective effects after ICH.

Article Snippet: Mitochondria were extracted using the Tissue and Cell Mitochondrial Extraction Kit (Beyotime, C3606, C3601) and separated from the cytoplasm through differential centrifugation of fresh brain tissue (processed within 1 h) and neuronal cells according to the manufacturer's instructions.

Techniques: Membrane, Activation Assay, Binding Assay

Journal: EMBO Molecular Medicine

Article Title: Mitochondrial damage drives T-cell immunometabolic paralysis after major surgery

doi: 10.1038/s44321-025-00324-1

Figure Lengend Snippet: ( A ) FCA of MitoTracker green and quantification in isolated mitochondria from CTL, n = 7 individual patients. ( B ) FCA of JC1 green and quantification of red/green fluorescence in isolated mitochondria from CTL, n = 4 individual patients. ( C ) Immunoblot of OXPHOS complexes using isolated mitochondria of CTL and normalized densitometric quantification of complex V, n = 5 individual patients. I–V complex I–V; H heat shock protein 60 (HSP60). Subunits detected by the antibody cocktail: complex I: subunit NDUFB8; complex II: subunit 30 kDa (SDHB); complex III: subunit Core 2 (UQCRC2); complex IV: subunit II (COXII); complex V: ATP synthase subunit alpha (ATP5). ( D , E ) Representative immunoblot of mitochondrial fusion and fission proteins of isolated mitochondria of CTL ( D ) and normalized densitometric quantification ( E ) of the respective proteins, n = 5/4/3 individual patients. ( F ) Representative projections of 3D image stacks of CTL stained with MitoTracker (MT, green, left) and the respective skeleton images (white, right), generated using mitochondrial analyzer, time points as indicated, representative of six individual patients. ( G ) Quantification of mitochondrial count per cell, n = 29 cells from three individual patients. ( H ) Quantification of mitochondrial length (left) and mean mitochondrial area using mitochondrial analyzer (right), n = 180/170 mitochondria from 14/22 cells from three individual patients (length) and n = 50 cells from three individual patients (area). ( I ) Representative projection of 3D image stacks (two per time point) of translocated mitochondria to the IS in CTL as indicated by MitoTracker (MT) Deep Red and CD3 FITC staining, and quantification of mitochondrial translocation to the CTL IS, time points as indicated, n = 14 from 5 individual patients. If not stated otherwise, data were represented as mean ± SEM. P values as indicated, paired t -test or Wilcoxon matched-pairs signed-rank test ( A – E ), and unpaired t -test or Mann–Whitney test ( G – I ), as appropriate. .

Article Snippet: Mitochondria Isolation Kit, Human , Miltenyi , 130-094-833.

Techniques: Isolation, Fluorescence, Western Blot, Staining, Generated, Translocation Assay, MANN-WHITNEY

Journal: EMBO Molecular Medicine

Article Title: Mitochondrial damage drives T-cell immunometabolic paralysis after major surgery

doi: 10.1038/s44321-025-00324-1

Figure Lengend Snippet: ( A ) FCA of JC1 and quantification of red/green fluorescence in CTL, n = 10 individual biological replicates. ( B ) Representative Seahorse OCR plot. ( C ) Pooled seahorse ECAR plot, mean ± SEM from ten individual biological replicates. ( D ) Fold change of basal OCR, maximum OCR, spare respiratory capacity (SRC) and ATP production, n = 10 individual biological replicates. ( E ) FCA of MitoTracker green and quantification in CTL, n = 10 individual biological replicates. ( F ) Immunoblot of mitochondrial fusion and fission proteins of isolated mitochondria of CTL and ( G ) normalized densitometric quantification of the respective proteins, n = 5/4/6 individual biological replicates. ( H ) FCA of Ki-67 and quantification in CTL, n = 11 individual biological replicates. ( I ) mRNA expression of IFNG , GZMB and PRF1 of CTL, relative to endogenous controls, n = 14/13/13 individual biological replicates. ( J ) Quantification of IFN-γ/ GZMB/ PRF1 secretion by CTL, n = 16/12/13 individual biological replicates. If not stated otherwise, data were represented as mean ± SEM. P values as indicated, one-sample t -test ( D ), paired t -test or Wilcoxon matched-pairs signed-rank test, as appropriate. .

Article Snippet: Mitochondria Isolation Kit, Human , Miltenyi , 130-094-833.

Techniques: Fluorescence, Western Blot, Isolation, Expressing

Journal: EMBO Molecular Medicine

Article Title: Mitochondrial damage drives T-cell immunometabolic paralysis after major surgery

doi: 10.1038/s44321-025-00324-1

Figure Lengend Snippet: ( A ) FCA of MitoTracker green and quantification in CD8 + CTL, n = 7 individual patients. ( B ) Representative Seahorse OCR plot. ( C ) Fold change of basal OCR, maximum OCR, spare respiratory capacity (SRC) and ATP production, n = 6 individual patients. ( D ) Quantification of mitochondrial length, n = 92 mitochondria from 28 cells from three individual patients. ( E ) Quantification of mitochondrial count per cell, n = 30/26 cells from three individual patients. ( F ) Representative confocal microscopy image of mitochondria translocated to the TCR in CTL as indicated by MitoTracker (MT) Deep Red and CD3 FITC staining, T2 image as depicted in Fig. , upper right image. ( G ) Quantification of mitochondrial translocation in proximity to the CTL IS, n = 12 from four individual patients. ( H ) Representative impedance plot and ( I ) quantification of relative CTL cytotoxicity using ECIS, n = 6 individual patients. If not stated otherwise, data are represented as mean ± SEM. P values as indicated, one-sample t -test ( C ), paired t -test or Wilcoxon matched-pairs signed-rank test ( A – C, H , I ), and unpaired t -test or Mann–Whitney test ( D – G ), as appropriate. .

Article Snippet: Mitochondria Isolation Kit, Human , Miltenyi , 130-094-833.

Techniques: Confocal Microscopy, Staining, Translocation Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: Photoactivated SOPP3 enables APEX2-mediated proximity labeling with high spatio-temporal resolution in live cells

doi: 10.1101/2024.06.23.595995

Figure Lengend Snippet: a Schematic of APEX2+SOPP3 mediated photoactivated-proximal labeling (photo-PL). B, Biotin. b Photosensitizer screening for APEX2 activation. Streptavidin blot showed photo-PL efficiency. anti-V5 and anti-HA indicated expression level of APEX2 and photosensitizers, respectively. Anti-α-tubulin, internal loading control for western blot. c , d Schematic ( c ) and construct designs ( d ) of APEX2+SOPP3 mediated PL to map proteome on ER-Mito contact sites. BP, Biotin Phenol. e Evaluation on the efficiency of photo-PL at ER-Mito contact sites. Anti-HA and anti-V5 indicated expression level of SOPP3-ERM and OMM-APEX2 respectively. f Left: confocal fluorescence imaging of APEX2+SOPP3 mediated photo-PL on ER-Mito contact sites. Outer membrane of mitochondria (OMM)-localized APEX2 and ER membrane (ERM)-localized SOPP3 were visualized by anti-V5 and anti-HA antibody. Biotinylation signals on the contact sites were visualized by Alexa Fluor 555-conjugated streptavidin. Illumination time, 5 seconds. Scale bar, 9 μm. Right: the ‘Surface’ tool in Imaris software was used to create a 3D rendering from each channel of confocal images of boxed region. Contact area algorithm was further performed to determine the interface (yellow) between ER (green) and Mito (cyan). Scale bar, 0.8 μm. g Validation on MAMs proteins from streptavidin-enriched PL samples. h , i Schematic and construct designs ( h ) and evaluation on photo-PL efficiency on cell-cell contact sites via western blotting analysis ( i ). Anti-V5 and anti-HA indicated expression level of APEX2-TM and SOPP3-TM respectively. j Confocal fluorescence imaging of APEX2+SOPP3 mediated photo-PL on cell-cell contact sites, stained with Alexa Fluore 555-conjugated streptavidin. Scale bar, 2 μm. Zoomed image from boxed region showed biotinylation labeling precisely on cell-cell contact sites. Scale bar, 1 μm. Illumination time, 10 seconds. k Schematic and construct design of chimeric APEX2-SOPP3-mediated photo-PL in ERM. l Evaluation on the efficiency of photo-PL mediated by chimeric APEX2-SOPP3 targeted to ERM via western blotting analysis. Anti-HA/Flag/V5 indicated expression level of chimeric APEX2-SOPP3-ERM, SOPP3-ERM and OMM-APEX2 respectively. m Confocal fluorescence imaging of photo-PL via APEX2-SOPP3 targeted to ERM. Chimeric APEX2-SOPP3-ERM and biotinylation signals was visualized by anti-HA antibody and Alexa Fluor 555-conjugated streptavidin respectively. Anti-Flag blot indicated expression level of chimeric APEX-SOPP3 targeted to various subcellular compartments. Illumination time, 5 seconds. Scale bar, 10 μm. n , o Schematic and construct designs ( n ) and evaluation on chimeric APEX2-SOPP3 on cell-cell contact sites via western blotting analysis ( o ). Anti-HA indicated expression level of chimeric APEX2-SOPP3-TM. p Confocal fluorescence imaging of chimeric APEX2-SOPP3 mediated photo-PL on cell surface. Chimeric APEX2-SOPP3 was visualized by anti-HA antibody. Biotinylation signals were visualized by Alexa Fluor 555-conjugated streptavidin. Scale bar, 8 μm. Illumination time, 5 seconds. q Flow cytometry analysis on p (30,000 cells per condition). After photo-PL, cells were stained with Alexa Fluor 555-conjugated streptavidin, followed with flow cytometry gating for quantification of biotinylation labeling on the surface of APEX2-SOPP3-TM cells (anti-HA positive, q up) and neighboring WT cells (anti-HA negative, q down). Blue light-activated SOPP3 showed more efficient to facilitate APEX2-mediated proximity labeling compared to that of 1 mM H 2 O 2 treatment. Illumination time, 10 seconds.

Article Snippet: Freshly isolated crude mitochondria from HeLa cells were resuspended in mitochondria storage buffer (Beyotime Biotechnology, C3601).

Techniques: Labeling, Activation Assay, Expressing, Control, Western Blot, Construct, Fluorescence, Imaging, Membrane, Software, Biomarker Discovery, Staining, Flow Cytometry

Journal: BMC Anesthesiology

Article Title: Propofol exerted the neuroprotective effects by regulating renin-angiotensin system and mitochondria-associated endoplasmic reticulum membrane in the postoperative cognitive dysfunction

doi: 10.1186/s12871-025-03503-2

Figure Lengend Snippet: Schematic diagram of the experimental protocol. The study was divided into two parts. A The in vivo study to establish a rat model of postoperative cognitive dysfunction (POCD). Aged Sprague-Dawley (SD) rats ( n = 6/group) underwent Morris Water Maze (MWM) training for 4 days (Pre-op Day 5 − 1) to establish a behavioral baseline, with a probe trial on Day − 1. On Day 0, splenectomy was performed under different anesthetics to induce POCD. Postoperative cognitive function was evaluated by MWM probe trials on Days 1, 3, 5, and 7. At the endpoint (Day 7), rats were euthanized for hippocampal tissue collection. Downstream analyses on hippocampal tissue included Long-Term Potentiation (LTP) recording, Renin-Angiotensin System (RAS) component ELISAs, and mitochondrial Reactive Oxygen Species (ROS) & ATP assays. B The in vitro mechanistic exploration. In Step 1, primary hippocampal neurons were isolated from P0 rat pups for cell culture. In Step 2, cultured neurons were treated with pharmacological agents such as Angiotensin II (Ang II), propofol, or siRNAs for 6 h. Downstream analyses on treated neurons included apoptosis assay (flow cytometry), Western blotting, mitochondria-associated membranes (MAMs) isolation, and transmission electron microscopy.

Article Snippet: Mitochondria were isolated from fresh hippocampal tissue using a Mitochondria Isolation Kit for Tissue (C3606, Beyotime, China) according to the manufacturer’s protocol.

Techniques: In Vivo, In Vitro, Isolation, Cell Culture, Apoptosis Assay, Flow Cytometry, Western Blot, Transmission Assay, Electron Microscopy

Journal: BMC Anesthesiology

Article Title: Propofol exerted the neuroprotective effects by regulating renin-angiotensin system and mitochondria-associated endoplasmic reticulum membrane in the postoperative cognitive dysfunction

doi: 10.1186/s12871-025-03503-2

Figure Lengend Snippet: Propofol reversed angiotensin II-induced inhibition of mitochondria-associated endoplasmic reticulum membrane formation. A Transmission electron microscopy was used to observe mitochondria-associated endoplasmic reticulum membranes. B Western blotting was used to detect the levels of mitochondria-associated endoplasmic reticulum membrane-related proteins, glucose-regulated protein 75, inositol 1,4,5-trisphosphate receptor type 3 and voltage-dependent anion channel 1. Mitochondrial calcium uptake ( C ), mitochondrial reactive oxygen species ( D ) and mitochondrial adenosine triphosphate ( E ) in each group. All data are presented as mean ± SD, with significance levels indicated as * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Mitochondria were isolated from fresh hippocampal tissue using a Mitochondria Isolation Kit for Tissue (C3606, Beyotime, China) according to the manufacturer’s protocol.

Techniques: Inhibition, Membrane, Transmission Assay, Electron Microscopy, Western Blot

Journal: Cell Reports Medicine

Article Title: A preclinical candidate of cyclophilin D inhibition improves alcohol-associated liver injury

doi: 10.1016/j.xcrm.2026.102654

Figure Lengend Snippet: Cyclophilin D (CypD) expression is upregulated in alcohol-associated liver disease (ALD) (A) Schematic representation of the chronic-binge ALD model (the NIAAA model). (B) Transmission electron microscopy analysis of liver mitochondria in healthy mice and ALD mice (left, mitochondrial swelling marked with a red arrow; scale bars, 2 μm [left panel]/500 nm [magnified part]); quantitative analysis of mitochondrial swelling area in the liver (right, n = 30 per group). (C) Quantitative PCR results of key mitochondrial functional proteins in the livers of healthy (control) mice and ALD mice ( n = 4 mice per group). (D) Representative western blot results of CypD protein in the livers of healthy mice and ALD mice; corresponding quantitative analyses were conducted ( n = 3 mice per group). (E) Representative western blot results of CypD protein in mouse liver tissues at different stages of steatosis; corresponding quantitative analysis was conducted ( n = 3 mice per group). (F) The expression level of Ppif mRNA in mouse liver tissues at different stages of steatosis ( n = 4 mice per group). (G) Expression changes of CypD protein in liver tissues at different stages of steatosis in a published clinical cohort (left panel, n = 12–36 participants per group). The expression of CypD protein is shown in liver tissue stratified by the absence of steatosis or inflammation (S/I 0), varying degrees of steatosis (S 1–3), and varying degrees of inflammatory activity (I 1–3) (right panel, n = 43–63 participants per group). S: grade of steatosis, I: grade of inflammation. S/I 0: no steatosis or inflammation; S 1–3: steatosis grade 1–3; I 1–3: inflammatory activity grade 1–3. (H) Representative result maps of CypD immunohistochemistry in liver tissues from healthy individuals and ALD patients (scale bars, 100 μm; n = 4 participants per group). Arrows indicate regions/cells with enhanced CypD immunoreactivity. (I and J) Single-cell sequencing data of Ppif expression in different liver cell types of humans. This set of data was downloaded from the Human Protein Atlas ( GSE115469 ). All data are presented as the means ± SDs. i.g., intragastric administration.

Article Snippet: Mitochondria isolation kit , MedChemExpress , Cat#HY-K1061.

Techniques: Expressing, Transmission Assay, Electron Microscopy, Real-time Polymerase Chain Reaction, Functional Assay, Control, Western Blot, Activity Assay, Immunohistochemistry, Single Cell, Sequencing

Journal: Cell Reports Medicine

Article Title: A preclinical candidate of cyclophilin D inhibition improves alcohol-associated liver injury

doi: 10.1016/j.xcrm.2026.102654

Figure Lengend Snippet: Hepatocyte-specific knockout of cyclophilin D (CypD) inhibits mPTP opening and improves hepatic mitochondrial dysfunction (A) Mitochondrial swelling, induced by calcium, was assessed in isolated liver mitochondria. The percentage decrease in initial optical density at 520 nm was used as a measure of swelling severity, determined by the slope of the resulting curve ( n = 5 mice per group). (B) The swelling degree of mitochondria in each group was analyzed by transmission electron microscopy (the mitochondrial swelling was marked by red arrows). Scale bars, 2 μm (upper panels) and 500 nm (zoom-out part). The swollen area of mitochondria in each group was quantitatively analyzed (right part; n = 30 per group). (C) Ca 2+ concentration in the liver tissues of indicated groups ( n = 5 mice per group). (D) Mitochondrial calcium retention capacity (CRC) was assessed in purified liver mitochondria from CypD f/f and CypD ΔHep mice using a Fluo-4 AM assay. Mitochondria were subjected to sequential 40 μM CaCl 2 pulses until reaching mPTP opening or calcium uptake saturation ( n = 5 mice per group). (E) Oxygen consumption rates (OCR) were measured in CypD f/f and CypD ΔHep mouse liver mitochondria. Mitochondrial respiration was challenged sequentially with oligomycin (2 μM), FCCP (2 μM), and rotenone (0.5 μM) ( n = 5 mice per group). (F) Quantification of state 3 respiration from (E) ( n = 5 mice per group). (G) The impact of calcium on mitochondrial membrane potential was examined in CypD f/f and CypD ΔHep mitochondria. (H) The maximal respiratory control ratio (RCR; state 3/state 4 respiration) was determined for isolated liver mitochondria from indicated groups ( n = 5 mice per group). (I) ADP/ATP ratio the in liver from of indicated groups ( n = 5 mice per group). (J) Mitochondrial ROS generation was detected in liver sections using MitoSox Red fluorescence. Scale bars, 100 μM (left). The ratio of MitoSox-red-stained area to DAPI-blue-stained area was quantified ( n = 5 mice per group) (right). All data are presented as the means ± SDs.

Article Snippet: Mitochondria isolation kit , MedChemExpress , Cat#HY-K1061.

Techniques: Knock-Out, Isolation, Transmission Assay, Electron Microscopy, Concentration Assay, Purification, Membrane, Control, Fluorescence, Staining

Journal: Cell Reports Medicine

Article Title: A preclinical candidate of cyclophilin D inhibition improves alcohol-associated liver injury

doi: 10.1016/j.xcrm.2026.102654

Figure Lengend Snippet: RN-0001 effectively alleviates alcohol-induced liver damage through CypD (A) Schematic diagram of the RN-0001 administration in the NIAAA chronic-binge ALD mouse model. (B) Changes in body weight and diet intake of mice from indicated groups ( n = 15 per group). (C) Changes in the levels of mouse serum biochemical parameters (ALT, AST, TG, and TC) in mice from indicated groups ( n = 3-5 per group). (D) Changes in mouse serum pro-inflammatory mediators (TNF-α and IL-6) levels from indicated groups ( n = 5 mice per group). (E) Liver weight and liver/body weight ratios were assessed in mice from indicated groups ( n = 5 mice per group). (F) Changes in mouse liver FFA and TG levels from indicated groups ( n = 5 mice per group). (G) Representative H&E and oil red O staining images of mouse liver sections from indicated groups are shown. Scale bars, 50 μm (left), NAFLD activity score (NAS score) and the measurement of oil red O staining (right, n = 4–5 mice per group). (H) Protein level changes in the liver from indicated groups (left) and their quantification ( n = 3 per group, right). (I) Degree of mitochondria swelling in each group analyzed by transmission electron microscopy (mitochondrial swelling marked by red arrows). Scale bars, 2 μm (upper panels) and 500 nm (zoom-out part). The swollen area of mitochondria in each group was quantitatively analyzed (right; n = 30 per group). (J) Changes of serum biochemical indexes (ALT, AST, TG, and TC) between CypD f/f and CypD ΔHep mice after ethanol and RN-0001 treatments ( n = 5 mice per group). (K) Representative H&E and oil red O staining images of liver sections of CypD f/f and CypD ΔHep mice after ethanol and RN-0001 treatments. Scale bars, 50 μm. (upper panels); NAFLD activity score (NAS score) and measurement of oil red O staining (below; n = 5 mice per group). (L) Degree of mitochondria swelling in CypD f/f and CypD ΔHep mice after ethanol and RN-0001 treatments analyzed by transmission electron microscopy. Scale bars, 2 μm (upper panels) and 500 nm (zoom-out part). The swollen area of mitochondria in each group was quantitatively analyzed (below; n = 30 per group). All data are presented as the means ± SDs. i.g., intragastric administration; i.p., intraperitoneal injection.

Article Snippet: Mitochondria isolation kit , MedChemExpress , Cat#HY-K1061.

Techniques: Staining, Activity Assay, Transmission Assay, Electron Microscopy, Injection

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Role of RGS12 in placental mitochondrial dysfunction and adverse pregnancy outcomes

doi: 10.1007/s00018-025-05999-w

Figure Lengend Snippet: RGS12 regulates tyrosine phosphorylation of ATP5B. A Whole-protein extracts (input) from HTR-8/SVneo cells, mouse 18.5dpc placenta, and human term placental tissues were subjected to immunoblotting using antibodies against Phosphotyrosine (p-Tyr) and β-actin prior to co-immunoprecipitation (co-IP). B-G Protein lysates from HTR-8/SVneo ( B and C ) cells, mouse 18.5dpc placenta ( D and E ), and human term placenta ( F and G ) tissues were incubated separately with anti-ATP5B, anti-Phosphotyrosine or control immunoglobulin G (IgG) antibodies for co-IP, and the bound proteins were subsequently detected by WB. RGS12 affects mitochondrial p-Tyr expression in HTR-8/SVneo cells ( H and I ). Stable transfections of HTR-8/SVneo cells were generated with knockdown (Control, shCTL; RGS12 knockdown, shRGS12) and overexpression (Control, CTL OE ; RGS12 overexpression, RGS12 OE ) of RGS12, and their mitochondrial and cytoplasmic proteins were extracted for WB detection of p-Tyr protein levels

Article Snippet: Following the manufacturer's protocol, a mitochondrial isolation kit (Novus Biologicals, US) was used to isolate mitochondria from placental tissue or trophoblast cells.

Techniques: Phospho-proteomics, Western Blot, Immunoprecipitation, Co-Immunoprecipitation Assay, Incubation, Control, Expressing, Transfection, Generated, Knockdown, Over Expression

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Role of RGS12 in placental mitochondrial dysfunction and adverse pregnancy outcomes

doi: 10.1007/s00018-025-05999-w

Figure Lengend Snippet: Knockdown of RGS12 leads to mitochondrial dysfunction in placental trophoblast cells. A WB analysis of RGS12 expression in the cytoplasm (Cyto) and mitochondria (Mito) when stable transfected with scramble control shRNA (shCTL) or shRGS12 in HTR-8/SVneo cells. B Representative HTR-8/SVneo cells transmission electron microscopy (TEM) images from each separate group (red arrows showed the swollen and damaged mitochondria). Quantitative measurement of ( B ), mitochondrial diameter ( C ), and mitochondrial area ( D ) in shCTL and shRGS12 groups using Image J software ( n = 3). E Mitochondrial DNA (mtDNA) copy number in stable shCTL and shRGS12-transfected HTR-8/SVneo cells as determined by PCR ( n = 5). F Relative ATP level in stable shCTL and shRGS12-transfected HTR-8/SVneo cells. G MitoTracker stained HTR-8/SVneo cells showing low intensity in shRGS12-transfected cells. Dihydroethidium stained HTR-8/SVneo cells ( G ) and fluorescence intensity quantification analysis ( H ) showing high ROS production in shRGS12-transfected HTR-8/SVneo cells relative to shCTL HTR-8/SVneo cells ( n = 3). I Total antioxidant capacity of stable shCTL and shRGS12-transfected HTR-8/SVneo cells measured by ABTS assay (n = 8). Values are expressed as expressed as means ± SEM, ** P < 0.01, *** P < 0.001 and **** P < 0.0001

Article Snippet: Following the manufacturer's protocol, a mitochondrial isolation kit (Novus Biologicals, US) was used to isolate mitochondria from placental tissue or trophoblast cells.

Techniques: Knockdown, Expressing, Transfection, Control, shRNA, Transmission Assay, Electron Microscopy, Software, Staining, Fluorescence, ABTS Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Role of RGS12 in placental mitochondrial dysfunction and adverse pregnancy outcomes

doi: 10.1007/s00018-025-05999-w

Figure Lengend Snippet: RGS12 knockout (KO) and placenta-specific RGS12 KO mice exhibit reduced birth weight and placental mitochondrial dysfunction. A Placenta derived from RGS12 flox/flox ; Elf5 -Cre or RGS12 flox/flox controls was subjected to immunohistochemistry with an antibody against mouse RGS12 (Scale bar, 50 μm). B-D Comparison of the weight ratio of fetal mice to placental weight ( B ) and comparison of placental weight ( C ) and fetal mouse weight ( D ) between the RGS12 flox/flox ; Elf5 -Cre and RGS12 flox/flox groups at 18.5 dpc ( n = 3). E Placenta derived from RGS12 KO or wild-type (WT) controls was subjected to immunohistochemistry with an antibody against mouse RGS12 (Scale bar, 50 μm). F–H Comparison of the weight ratio of fetal mice to placental weight ( F ) and comparison of placental weight ( G ) and fetal mouse weight ( H ) between the RGS12 KO and WT groups at 18.5 dpc ( n = 5). (mating between RGS12 KO females and RGS12 KO males). I Representative immunoblots for mitochondrial protein p-Tyr in 18.5 dpc placenta from RGS12 flox/flox ; Elf5 -Cre and RGS12 flox/flox groups. Notably, the p-Tyr level in RGS12 flox/flox mice was higher than in RGS12 flox/flox ; Elf5 -Cre mice. J Representative transmission electron microscopy (TEM) images of 18.5dpc placentas from RGS12 flox/flox ; Elf5-Cre and RGS12 flox/flox mice. Red arrows indicate mitochondria. K-M Comparison of mitochondrial DNA copy number ( K ) ( n = 7), total antioxidant capacity ( L ) ( n = 5), and ATP content ( M ) ( n = 6) in the placenta of mice at 18.5 dpc in the RGS12 flox/flox ; Elf5 -Cre and RGS12 . flox/flox groups. Values are expressed as means ± SEM, * P < 0.05 and ** P < 0.01

Article Snippet: Following the manufacturer's protocol, a mitochondrial isolation kit (Novus Biologicals, US) was used to isolate mitochondria from placental tissue or trophoblast cells.

Techniques: Knock-Out, Derivative Assay, Immunohistochemistry, Comparison, Western Blot, Transmission Assay, Electron Microscopy

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Role of RGS12 in placental mitochondrial dysfunction and adverse pregnancy outcomes

doi: 10.1007/s00018-025-05999-w

Figure Lengend Snippet: Restoring RGS12 expression reverses the abnormal hormonal and mitochondrial phenotype. A Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) was employed to evaluate the transfection efficiency of RGS12 OE in RGS12 knockdown HTR-8/SVneo cells (shCTL-CTL OE and shRGS12-RGS12 OE group) ( n = 10). B-H Cell supernatants from shCTL-CTL OE and shRGS12-RGS12 OE -transfected HTR-8/SVneo cells were subjected to ELISA analysis for P ( B ), E2 ( C ), E2/P ( D ), SOD ( E ), GSH ( F ), MDA ( G ), NO ( H ) ( n = 9 and 12). I-K Comparison of mitochondrial DNA copy number ( I ), ATP content ( J ), and total antioxidant capacity ( K ) ( n = 5 and 6) in the shCTL-CTL OE and shRGS12-RGS12 OE -transfected HTR-8/SVneo cells. Values are expressed as means ± SEM, ns, not significant

Article Snippet: Following the manufacturer's protocol, a mitochondrial isolation kit (Novus Biologicals, US) was used to isolate mitochondria from placental tissue or trophoblast cells.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Transfection, Knockdown, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Role of RGS12 in placental mitochondrial dysfunction and adverse pregnancy outcomes

doi: 10.1007/s00018-025-05999-w

Figure Lengend Snippet: Decreased RGS12 Expression and Mitochondrial Protein Phosphorylation in PTB Human Placentas and Reduced Hormone Levels in Peripheral Blood. A and B WB detection of RGS12 expression levels in PTB human placentas ( A ) and quantification analysis using Image J ( B ) ( n = 6). C RT–qPCR detection of RGS12 transcript levels in PTB human placentas ( n = 10). D WB analysis of phosphorylation levels of cytoplasmic and mitochondrial proteins in PTB human placentas, with β-actin and HSP60 used as internal references, respectively. E and F Comparison of ATP content ( E ) and mitochondrial DNA copy number ( F ) in the TB and PTB human placentas ( n = 3). G-J ELISA assessment of P ( G ), E2 ( H ), E2/P ( I ) ( n = 6), and NO ( J ) ( n = 3) levels in TB and PTB human placentas. Values are expressed as means ± SEM, * P < 0.05 and ** P < 0.01, ns, not significant

Article Snippet: Following the manufacturer's protocol, a mitochondrial isolation kit (Novus Biologicals, US) was used to isolate mitochondria from placental tissue or trophoblast cells.

Techniques: Expressing, Phospho-proteomics, Quantitative RT-PCR, Comparison, Enzyme-linked Immunosorbent Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Role of RGS12 in placental mitochondrial dysfunction and adverse pregnancy outcomes

doi: 10.1007/s00018-025-05999-w

Figure Lengend Snippet: Schematic diagram of the molecular mechanisms underlying RGS12-mediated regulation of placental mitochondrial function in preterm birth. Under physiological conditions, RGS12 associates with ATP5B and activates p-Tyr of ATP5B to regulate mitochondrial homeostasis, maintaining normal placental function until full-term delivery. Various risk factors such as LPS and PGE2 decrease RGS12 expression, reducing ATP synthesis activity and antioxidant capacity, thereby increasing ROS levels. Additionally, the lack of RGS12 results in decreased mitochondrial DNA copy number and mitochondrial dysfunction. Finally, mitochondrial dysfunction may lead to placental dysfunction and PTB by activating the p38MAPK signaling pathway and reducing stress tolerance

Article Snippet: Following the manufacturer's protocol, a mitochondrial isolation kit (Novus Biologicals, US) was used to isolate mitochondria from placental tissue or trophoblast cells.

Techniques: Expressing, Activity Assay