Journal: The Journal of Cell Biology

Article Title: Long-lived mitochondrial cristae proteins in mouse heart and brain

doi: 10.1083/jcb.202005193

Figure Lengend Snippet: Mitochondrial proteins are exceptionally long lived in tissues containing enduring postmitotic cells. (A) Whole-animal metabolic pulse 15 N labeling scheme to identify and measure LLPs (≥4 mo) in mice. (B) Analytical workflow illustrating how the dissected tissues were homogenized and digested to peptides, and 15 N and 14 N peptides were identified and quantitated with LC-MS/MS–based proteomics. (C) Summary of protein identifications. Left plot indicates the number of 15 N proteins identified (i.e., new, ≤4 mo old), right plot indicates 14 N proteins identified (i.e., ≥4 mo old). (D) Tissues enriched with long-lived cells selectively harbor LLPs despite a similar number of total proteins identified in most tissues. (E) GO analysis of the LLPs in tissues that contain enduring postmitotic cells revealed that terms related to mitochondria (mito.) are significantly enriched. syn., synthesis; trans., transport. Data in C and D are mean ± SEM; in C–E, n = three mice for each tissue analyzed. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by Kruskal–Wallis ANOVA with Tukey’s multiple comparisons test. See also and .

Article Snippet: For experiments requiring purified mitochondria, indicated tissues were thawed on ice, and mitochondria were extracted according to the Miltenyi Biotec Mitochondria Extraction–Tissue Isolation Protocol (130-097-340).

Techniques: Labeling, Liquid Chromatography with Mass Spectroscopy

Journal: The Journal of Cell Biology

Article Title: Long-lived mitochondrial cristae proteins in mouse heart and brain

doi: 10.1083/jcb.202005193

Figure Lengend Snippet: LLPs are preferentially associated with the IMM. (A) Schematic illustrating mitochondrial subcompartmentalization and relevant protein complexes. (B) Workflow to immune-capture mitochondria. Cortex, heart, and spleen from 15 N-pulsed mice were homogenized using a gentleMACS system, and dissociated mitochondria were purified by immuno-capture using anti-Tom20 antibodies (Miltenyi Biotec). Purified mitochondria were analyzed using LC-MS/MS, mitochondrial proteins were filtered using MitoCarta 3.0, and mt-LLPs were grouped and examined based on their submitochondrial localization and Mitochondrial Pathway assignments. FT, flow through; IC, immunocapture; IBM, inner boundary membrane. (C) The total number of mitochondrial proteins identified based on 15 N and 14 N MS/MS spectral matches per tissue (blue) along with the number of identified 14 N mt-LLPs (tan). mt-LLPs are enriched at IMM in both heart and cortical extracts, but not in the other subcompartments. None of the mitochondrial proteins in spleen were identified as mt-LLPs. (D) MitoPathway analysis of mt-LLPs. mt-LLPs are enriched in several protein complexes including OXPHOS, Tim22, TOM, SAM, and the cristae formation (i.e., MICOS complex). BR, biological replicate. Data in C are mean ± SEM; for C and D, n = 3 mice for cortex and heart, 2 for spleen. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 by Kruskal–Wallis ANOVA with Tukey’s multiple comparisons test. See also and .

Article Snippet: For experiments requiring purified mitochondria, indicated tissues were thawed on ice, and mitochondria were extracted according to the Miltenyi Biotec Mitochondria Extraction–Tissue Isolation Protocol (130-097-340).

Techniques: Purification, Liquid Chromatography with Mass Spectroscopy, Membrane, Tandem Mass Spectroscopy

Journal: The Journal of Cell Biology

Article Title: Long-lived mitochondrial cristae proteins in mouse heart and brain

doi: 10.1083/jcb.202005193

Figure Lengend Snippet: LLPs are preferentially associated with the IMM. Related to . (A) Coverage of mitochondrial proteome across immuno-isolated mitochondria. Cumulative number of proteins identified shown. n = 2–4 mice. (B) Immuno-isolated mitochondria are substantially enriched in mitochondrial proteins. n = 4 mice. (C) Submitochondrial distribution of identified mitochondrial proteins from cortical, heart, and spleen tissue extracts. Identified proteins are shown in orange; MitoCarta3.0 proteins are shown in gray. n = 3 mice. (D) Schematic illustrating two-generation whole-animal metabolic 15 N-dynamic pulse compared with 14 N pulse-chase experiment design. (E) Venn diagram comparing proteins identified in the 15 N-dynamic pulse and 14 N pulse-chase experiments. enrich., enrichment. (F) GO analysis of the mt-LLPs identified after 4 mo of 14 N pulse-chase showing an enrichment for terms related to mitochondria. See also . (G) Time course pulse-chase experiment of 15 N-labeled mice (born to 15 N-labeled females) that were chased with 14 N diet for 0, 2, 4, and 6 mo. Note that the 4-mo 14 N pulse chase time point shown here represents the same dataset as in panels D and E. (H) Metabolic labeling efficiency of second-generation pups analyzed at t = 0 (at weaning) in the 14 N pulse-chase experiments. Average peptide labeling percentage is indicated ± SD. (I) Summary of protein degradation trends across chase periods. Only proteins identified at t = 0 which were then tracked through the time course are shown. n = 3–4 mice.

Article Snippet: For experiments requiring purified mitochondria, indicated tissues were thawed on ice, and mitochondria were extracted according to the Miltenyi Biotec Mitochondria Extraction–Tissue Isolation Protocol (130-097-340).

Techniques: Isolation, Pulse Chase, Labeling

Journal: The Journal of Cell Biology

Article Title: Long-lived mitochondrial cristae proteins in mouse heart and brain

doi: 10.1083/jcb.202005193

Figure Lengend Snippet: Long-lived subunits of OXPHOS complexes integrate into individual and higher-order complexes. (A) Solubilized cortical mitochondria extracts isolated from 15 N-pulsed mice separated by BN-PAGE to resolve mitochondrial complexes and supercomplexes. Representative Coomassie (Coom.)-stained BN-PAGE gel and Western blot probed with an OXPHOS antibody cocktail. Mitochondrial complexes and supercomplexes are annotated. (B and C) 40 gel slices excised from each gel lane were subjected to in-gel trypsin digestion and LC-MS/MS analysis. FA values were calculated for each protein in a given complex and plotted. C.I, complex I; C.II, complex II; C.III, complex III; C.IV, complex IV; CV m , complex V monomer; CV d , complex V dimer; CV o , complex V oligomer; SC, supercomplex. Data points in B and C, n = 4 mice; mean ± SEM.

Article Snippet: For experiments requiring purified mitochondria, indicated tissues were thawed on ice, and mitochondria were extracted according to the Miltenyi Biotec Mitochondria Extraction–Tissue Isolation Protocol (130-097-340).

Techniques: Isolation, Staining, Western Blot, Liquid Chromatography with Mass Spectroscopy

Journal: The Journal of Cell Biology

Article Title: Long-lived mitochondrial cristae proteins in mouse heart and brain

doi: 10.1083/jcb.202005193

Figure Lengend Snippet: Limited mixing and exchange of mt-LLPs. (A) Schematic illustrating DSSO cross-linking of mitochondria isolated from 15 N-pulse–labeled heart tissue extracts, sample processing, and MS1/MS2/MS3 analysis. (B) Silver-stained gel of control and cross-linked heart mitochondrial isolations. DSSO was added at a concentration of 1 mM; control was treated with DMSO only. Overview of cross-links between different mitochondrial proteins identified in the heart. (C) Number of cross-links identified in heart mitochondria according to the cross-link type. (D) Number of cross-links identified in heart mitochondria according to the peptide isotope build. (E) Map of the mitochondrial protein cross-links. 15 N- 15 N (new-new) peptide cross-links are shown in blue; 14 N- 14 N (old-old) peptide cross-links are shown in yellow; mixed 15 N- 14 N (new-old) peptide cross-links are shown in green. (F) Summary of identified cross-linked peptides broken down by cross-link type and nitrogen isotype. Maps illustrating cross-linked peptides according to nitrogen isotype. In B, E, and F, maps were generated in XiView proteins categorized based on MitoCarta3.0. Data in C, D, and F are mean ± SEM; n = 2 mice. *, P < 0.05; **, P < 0.01, by Kruskal–Wallis ANOVA with Tukey’s multiple comparisons test. See also .

Article Snippet: For experiments requiring purified mitochondria, indicated tissues were thawed on ice, and mitochondria were extracted according to the Miltenyi Biotec Mitochondria Extraction–Tissue Isolation Protocol (130-097-340).

Techniques: Isolation, Labeling, Staining, Control, Concentration Assay, Generated

Journal: Cell Death & Disease

Article Title: Mitochondrial transplantation: adaptive bio-enhancement

doi: 10.1038/s41419-025-07643-8

Figure Lengend Snippet: A Analysis of membrane potential in both cells and animals isolated mitochondria. Ctrl: Mitochondria were subjected to two freeze-thaw cycles at −80 °C and 37 °C to completely disrupt their structure and function, serving as a comparison, n = 3. B ATP production capacity in both cells and animals isolated mitochondria. Ctrl: Mitochondria were subjected to two freeze-thaw cycles at −80 °C and 37 °C, used for comparison, n = 3. C Mitochondria were randomly selected from cells (Vero) and animals (Bull Frog) for electron microscopy to observe their morphology. Scale bar: 500 nm. D – G Activity of respiratory chain complexes I and III in both cells and animals isolated mitochondria, n = 3. H Co-localization fluorescence imaging at 24 h of transplanted mitochondria labeled with Mito-Tracker Green (green fluorescence) from Eel, MDCK, CRFK, Sparrow, Vero, and Lizard, alongside AC16, L929, and HepG2 cells labeled with WGA594 (red fluorescence). Blue fluorescence (DAPI) represents the nucleus. Scale bar: 10 μm. I – N Immunoreactivity of IL-6, IL-10, and TNF-α in cell culture medium collected 24 h after the transplantation of exogenous mitochondria, n = 3. Ctrl: Untreated group. ns no statistical significance, **** p < 0.0001.

Article Snippet: Mitochondria were isolated from cultured cells, animal tissues, and plant tissues using the Mitochondria-Cytosol Protein Isolation Kit (Applygen) and the Plant Mitochondrial Extraction Kit (Solarbio).

Techniques: Membrane, Isolation, Comparison, Electron Microscopy, Activity Assay, Fluorescence, Imaging, Labeling, Cell Culture, Transplantation Assay

Journal: Cell Death & Disease

Article Title: Mitochondrial transplantation: adaptive bio-enhancement

doi: 10.1038/s41419-025-07643-8

Figure Lengend Snippet: A , I ATP production capacity in isolated mitochondria. Ctrl: Mitochondria subjected to two freeze-thaw cycles at −80 °C and 37 °C, used for comparison, n = 3. B , J Membrane potential in isolated mitochondria. Ctrl: Mitochondria subjected to two freeze-thaw cycles at −80 °C and 37 °C, used for comparison, n = 3. C Effects of mitochondrial transplantation from four different species on the viability of BMDM cells after H 2 O 2 treatment. Ctrl: Untreated group, n = 3. D Mitochondrial ROS fluorescence intensity in BMDM cells 24 h after transplantation of mitochondria from four different species, followed by H 2 O 2 treatment. Ctrl: Untreated group, n = 3. E , F Effects of mitochondrial transplantation from four different species on MDA and GSH levels in BMDM cells after H 2 O 2 treatment. Ctrl: Untreated group, n = 3. G , H Fluorescence imaging and statistical analysis of the internalization of mitochondria A labeled with Mito-Tracker Red and mitochondria B labeled with Mito-Tracker Green in BMDM cells after H 2 O 2 treatment. Original image scale bar: 10 µm; magnified region scale bar: 2 µm, n = 9. K Effects of mitochondrial transplantation from four different species on the viability of AC16 cells after CCCP treatment. Ctrl: Untreated group, n = 3. L – N Changes in IL-6, IL-10, and TNF-α levels in AC16 cells after CCCP treatment, following mitochondrial transplantation from four different species. Ctrl: Untreated group, n = 3. ns no statistical significance, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Mitochondria were isolated from cultured cells, animal tissues, and plant tissues using the Mitochondria-Cytosol Protein Isolation Kit (Applygen) and the Plant Mitochondrial Extraction Kit (Solarbio).

Techniques: Isolation, Comparison, Membrane, Transplantation Assay, Fluorescence, Imaging, Labeling

Journal: Cell Death & Disease

Article Title: Mitochondrial transplantation: adaptive bio-enhancement

doi: 10.1038/s41419-025-07643-8

Figure Lengend Snippet: A Co-localization fluorescence imaging 24 h after the fusion of HL1 cells labeled with WGA647 and H9C2 cells labeled with WGA594. Scale bar: 20 µm. B Flow cytometric sorting of hybrid cells 24 h after the fusion of HL1 cells labeled with WGA647 and H9C2 cells labeled with WGA488. C , D Western blot analysis of the mitochondrial fusion and fission proteins Mfn1, Opa1, and Drp1 in HL1, H9C2, and HL1 + H9C2 cells on the second day after cell fusion, n = 3. E ATP production capacity in isolated mitochondria, Ctrl: Mitochondria subjected to two freeze-thaw cycles at −80 °C and 37 °C, used for comparison, n = 3. F JC-1 membrane potential detection of isolated mitochondria. Ctrl: Mitochondria subjected to two freeze-thaw cycles at −80 °C and 37 °C, used for comparison, n = 3. G Effects of transplantation of three different types of mitochondria on the viability of AC16 cells after H 2 O 2 treatment. Ctrl: Untreated group, n = 3. H Effects of transplantation of three different types of mitochondria on mitochondrial ROS levels in AC16 cells after H 2 O 2 treatment. Ctrl: Untreated group, n = 3. I , J Effects of transplantation of mitochondria from three different species on MDA and GSH levels in AC16 cells after H 2 O 2 treatment. Ctrl: Untreated group, n = 3. ns no statistical significance, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Mitochondria were isolated from cultured cells, animal tissues, and plant tissues using the Mitochondria-Cytosol Protein Isolation Kit (Applygen) and the Plant Mitochondrial Extraction Kit (Solarbio).

Techniques: Fluorescence, Imaging, Labeling, Western Blot, Isolation, Comparison, Membrane, Transplantation Assay

Journal: Cell Death & Disease

Article Title: Mitochondrial transplantation: adaptive bio-enhancement

doi: 10.1038/s41419-025-07643-8

Figure Lengend Snippet: A , H ATP production capacity in isolated mitochondria. Ctrl: Mitochondria subjected to two freeze-thaw cycles at −80 °C and 37 °C, used for comparison, n = 3. B , I Membrane potential in isolated mitochondria. Ctrl: Mitochondria subjected to two freeze-thaw cycles at −80 °C and 37 °C, used for comparison, n = 3. C Effects of mitochondria transplantation from four different species on the viability of HepG2 cells after CCCP treatment. Ctrl: Untreated group, n = 3. D Effects of mitochondria transplantation from four different species on mitochondria ROS in HepG2 cells after CCCP treatment. Ctrl: Untreated group, n = 3. E Detection of DNA damage repair in HepG2 cells after CCCP treatment following mitochondria transplantation from four different species. Ctrl: Untreated group, n = 3. F , G Fluorescence imaging and statistical analysis of the internalization of Lizard mitochondria labeled with Mito-Tracker Deep-Red and Bull Frog mitochondria labeled with Mito-Tracker Green in HepG2 cells after CCCP treatment. Original image scale bar: 10 µm, magnified region scale bar: 2 µm, n = 6. J Effects of mitochondria transplantation from four different species on the viability of BMDM cells after LPS treatment. Ctrl: Untreated group, n = 3. K – M Changes in IL-6, IL-10, and TNF-α levels in BMDM cells after LPS treatment following mitochondria transplantation from four different species. Ctrl: Untreated group, n = 3. N Detection of DNA damage repair in BMDM cells after LPS treatment following mitochondria transplantation from four different species, n = 3. O , P Fluorescence imaging and statistical analysis of the internalization of Lizard mitochondria labeled with Mito-Tracker Green, Sparrow mitochondria labeled with Mito-Tracker Deep-Red, and Salmon mitochondria labeled with Mito-Tracker Red in BMDM cells after LPS treatment. Original image scale bar: 10 µm, magnified region scale bar: 5 µm, n = 6. ns no statistical significance, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Mitochondria were isolated from cultured cells, animal tissues, and plant tissues using the Mitochondria-Cytosol Protein Isolation Kit (Applygen) and the Plant Mitochondrial Extraction Kit (Solarbio).

Techniques: Isolation, Comparison, Membrane, Transplantation Assay, Fluorescence, Imaging, Labeling

Journal: Cell Death & Disease

Article Title: Mitochondrial transplantation: adaptive bio-enhancement

doi: 10.1038/s41419-025-07643-8

Figure Lengend Snippet: A , B Evaluation of isolated mitochondrial functions through ATP production and mitochondrial membrane potential (MMP) assays, with mitochondria subjected to two freeze-thaw cycles at −80 °C and 37 °C to completely disrupt their structure and function as a control, n = 3. C Biodistribution of exogenous mitochondria in the mouse liver, kidney, and heart detected by in vivo imaging 24 h after intravenous tail vein injection of mitochondria. The blank group consists of untreated LPS-Ctrl group mice. D , E Assessment of motor function in acute inflammation model mice and normal model mice 3 days after the transplantation of two different types of mitochondria, with untreated groups as control, n = 6. F – H Changes in body weight of acute inflammation model mice and normal model mice before and 1 week after the transplantation of two different types of mitochondria, with untreated groups as control, n = 6. I Grip strength measurements in acute inflammation model mice and normal model mice 3 days after the transplantation of two different types of mitochondria, with untreated groups as control, n = 6. ns no statistical significance, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Mitochondria were isolated from cultured cells, animal tissues, and plant tissues using the Mitochondria-Cytosol Protein Isolation Kit (Applygen) and the Plant Mitochondrial Extraction Kit (Solarbio).

Techniques: Isolation, Membrane, Control, In Vivo Imaging, Injection, Transplantation Assay

Journal: Cell Death & Disease

Article Title: Mitochondrial transplantation: adaptive bio-enhancement

doi: 10.1038/s41419-025-07643-8

Figure Lengend Snippet: A – C Changes in serum levels of IL-6, TNF-α, and IL-10 in acute inflammation model mice and normal model mice 1 week after transplantation of two different mitochondria, with untreated groups as control, n = 3. D – F Evaluation of kidney and liver function via serum Cr, ALT, and AST levels in acute inflammation model mice and normal model mice 1 week after transplantation of two different mitochondria, with untreated groups as control, n = 3. G , H Measurement of serum MDA levels and SOD activity in acute inflammation model mice and normal model mice 1 week after transplantation of two different mitochondria, with untreated groups as control, n = 3. I – K Analysis of JNK and P-JNK, p65, and P-p65 protein expression levels in liver and kidney tissues via Western blot 1 week after transplantation of two different mitochondria in acute inflammation model mice and normal model mice, with untreated groups as control, n = 3. L Effects of two different mitochondria on the viability of LPS-treated BMDM cells 24 h after transplantation, with untreated groups as control, n = 3. M Quantitative analysis of mitochondrial ROS levels in LPS-treated BMDM cells 24 h after transplantation of two different mitochondria, with untreated groups as control, n = 3. ns no statistical significance, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Mitochondria were isolated from cultured cells, animal tissues, and plant tissues using the Mitochondria-Cytosol Protein Isolation Kit (Applygen) and the Plant Mitochondrial Extraction Kit (Solarbio).

Techniques: Transplantation Assay, Control, Activity Assay, Expressing, Western Blot

Journal: Stem Cell Research & Therapy

Article Title: Adipose-derived stem cells attenuate rheumatoid arthritis by restoring CX 3 CR1 + synovial lining macrophage barrier

doi: 10.1186/s13287-025-04144-5

Figure Lengend Snippet: Mitochondrial transfer from ADSCs during the RA treatment process. A-E used CX 3 CR1 − synovial macrophages and F used CX 3 CR1 + synovial macrophages. (A) Transfer of mitochondria from WT-ADSCs (no fluorescence) stained with MitoTracker-Green to CX 3 CR1 − synovial macrophages in vitro. Scale bar, 20 μm. (B) . Transfer of mitochondria from EGFP-ADSCs (EGFP fluorescence) stained with MitoTracker-Red to CX 3 CR1 − synovial macrophages in vitro. Scale bar, 20 μm. (C) Inhibition of mitochondrial transfer from ADSCs by pretreatment with 10 µM nocodazole for 6 h. Scale bar, 40 μm. (D) Red fluorescence intensity of single CX 3 CR1 − lining macrophage in different groups. (E) Mitochondrial transfer from EGFP-Cox8-ADSCs to CX 3 CR1 − macrophages. Scale bar, 15 μm. (F) Mitochondrial transfer from EGFP-Cox8-ADSCs to CX 3 CR1 + lining macrophages. Scale bar, 30 & 10 μm, from left to right. (G) CLSM observation of mitochondrial transfer from EGFP-Cox8-ADSCs to synovial cells in vivo on STA day 4. Scale bar, 20 μm. (H) Isolated mitochondria of ADSCs stained with MitoTracker-Green. Scale bar, 100 μm. (I) RA clinical score changes when the TNTs of ADSCs were inhibited using nocodazole. (J) The role of the mitochondrial transplantation from ADSCs in RA treatment

Article Snippet: Mitochondria extraction was conducted using mitochondrial extraction kit (Solarbio, China) and the isolated mitochondria were labeled with MitoTracker-Green for observation.

Techniques: Fluorescence, Staining, In Vitro, Inhibition, In Vivo, Isolation, Transplantation Assay

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