Journal: JACC: Basic to Translational Science

Article Title: c-FLIP Protects Cardiac Microcirculation in Sepsis-Induced Myocardial Dysfunction Via FUNDC1-Mediated Regulation of Mitochondrial Autophagy

doi: 10.1016/j.jacbts.2025.02.016

Figure Lengend Snippet: Construction of In Vitro Model and Observation of Mitochondrial Autophagy and Expression Changes of c-FLIP, JNK, and FUNDC1 (A and B) Optical microscopy of cardiac microvascular endothelial cell (CMEC) morphology and CCK-8 assay for cell proliferation after 12 hours of different lipopolysaccharide (LPS) concentrations. (C and D) Optical microscopy of CMECs morphology and CCK-8 assay for cell proliferation at various times after 10 μg/mL LPS treatment. (E and F) c-FLIP expression at various times after 10 μg/mL LPS treatment. (G) Transmission electron microscopy (×12,000) of CMECs microstructure to assess mitochondrial damage and autophagy (representative images). (H to L) Western blot of mitochondrial autophagy-related proteins. (M-Q) Expression of c-FLIP, total c-Jun N-terminal kinase (t-JNK), phosphorylated JNK (p-JNK), total Fun14 domain-containing protein 1 (t-FUNDC1), phosphorylated FUNDC1 (p-FUNDC1) proteins, and FUNDC1 mRNA. Note: n = 3/group; 3 technical replicates; mean ± SEM; Student's t -test (N-Q), analysis of variance with Tukey’s post hoc test (B, D, F, and I to L). ∗ P < 0.05 vs Ctrl (control) group, ∗∗ P < 0.01 vs Ctrl group, ∗∗∗ P < 0.001 vs Ctrl group. Abbreviations as in .

Article Snippet: The samples were then incubated overnight at 4 °C with primary antibodies, including: CD34 (Abcam, ab81289, 1:100), c-FLIP (Affinity Biosciences, DF7010, 1:100), caspase-3 (Proteintech, 66470-2-Ig, 1:100), caspase-9 (Proteintech, 66169-1-Ig, 1:100), endothelial nitric oxide synthase (eNOS) (Santa Cruz Biotechnology, Sc-376751, 1:100), endothelin 1 (ET-1) (Novus Biologicals, NB300-526, 1:100), vascular endothelial cadherin (VE-cadherin) (Santa Cruz Biotechnology, sc-9989, 1:100), t-FUNDC1 (Bioss Antibodies, Bs-13227R, 1:100), and p-JNK (Santa Cruz Biotechnology, sc-6254, 1:100).

Techniques: In Vitro, Expressing, Microscopy, CCK-8 Assay, Transmission Assay, Electron Microscopy, Western Blot, Control

Journal: JACC: Basic to Translational Science

Article Title: c-FLIP Protects Cardiac Microcirculation in Sepsis-Induced Myocardial Dysfunction Via FUNDC1-Mediated Regulation of Mitochondrial Autophagy

doi: 10.1016/j.jacbts.2025.02.016

Figure Lengend Snippet: Changes in Mitochondrial Autophagy Activity at Different Stages of LPS-Induced Injury and the Regulatory Role of c-FLIP (A) Electron microscopy of mitochondrial structure and autophagy in CMECs 12 hours post–LPS-induced injury. Yellow arrows indicate damage to mitochondrial structure; green arrows indicate autophagosomes or autolysosomes. (B to F) Western blot analysis of the expression of proteins related to mitochondrial autophagy in CMECs 12 hours after LPS-induced injury. (G-L) Western blot analysis of the expression of mitochondrial autophagy-related proteins in CMECs 36 hours post–LPS-induced injury. (M and N) Immunofluorescence used to detect the fusion of mitochondria and lysosomes. Green fluorescence stains mitochondria, red fluorescence stains lysosomes, blue fluorescence stains nuclei, and orange-yellow fluorescence indicates the fusion of mitochondria and lysosomes, representing mitochondrial autophagy. Note: n = 3/group; 3 technical replicates; mean ± SEM; analysis of variance with Tukey’s post hoc test. Ad-c-FLIP was used to infect CMECs to overexpress the c-FLIP protein; siRNA-FUNDC1 was applied to silence the FUNDC1 gene, thereby reducing or preventing the production of FUNDC1 protein. ∗ P < 0.05 vs Ctrl group, # P < 0.05 vs LPS + Ad-NC CMECs group, @ P < 0.05 vs LPS + Ad-c-FLIP CMECs group. Abbreviations as in , , , and .

Article Snippet: The samples were then incubated overnight at 4 °C with primary antibodies, including: CD34 (Abcam, ab81289, 1:100), c-FLIP (Affinity Biosciences, DF7010, 1:100), caspase-3 (Proteintech, 66470-2-Ig, 1:100), caspase-9 (Proteintech, 66169-1-Ig, 1:100), endothelial nitric oxide synthase (eNOS) (Santa Cruz Biotechnology, Sc-376751, 1:100), endothelin 1 (ET-1) (Novus Biologicals, NB300-526, 1:100), vascular endothelial cadherin (VE-cadherin) (Santa Cruz Biotechnology, sc-9989, 1:100), t-FUNDC1 (Bioss Antibodies, Bs-13227R, 1:100), and p-JNK (Santa Cruz Biotechnology, sc-6254, 1:100).

Techniques: Activity Assay, Electron Microscopy, Western Blot, Expressing, Immunofluorescence, Fluorescence

Journal: JACC: Basic to Translational Science

Article Title: c-FLIP Protects Cardiac Microcirculation in Sepsis-Induced Myocardial Dysfunction Via FUNDC1-Mediated Regulation of Mitochondrial Autophagy

doi: 10.1016/j.jacbts.2025.02.016

Figure Lengend Snippet: Mitochondrial Function Changes in the Late Stage of LPS-Induced Injury and the Protective Role of c-FLIP (A and B) Measurement of mitochondrial Ca 2+ levels using the cell-permeant Ca 2+ fluorescent probe Rhod-2 AM, quantified by flow cytometry to reflect Ca 2+ levels inside mitochondria. (C) Quantification of mitochondrial reactive oxygen species (ROS) using the DCFH-DA probe. (D) Measurement of mitochondrial adenosine triphosphate (ATP) content by assessing absorbance. (E and F) Assessment of the mitochondrial permeability transition pore (mPTP) state using the Calcein AM fluorescent probe, with CoCl 2 quenching fluorescence in non-mitochondrial regions; changes in mitochondrial fluorescence intensity are analyzed via flow cytometry, where a decrease in fluorescence indicates mPTP opening. (G and H) Analysis of changes in mitochondrial membrane potential (ΔΨm) using the JC-1 fluorescent probe, with flow cytometry used to analyze the ratio of red to green fluorescence for quantitative assessment. Note: n = 3/group; 3 technical replicates; mean ± SEM; analysis of variance with Tukey’s post-hoc test. Ad-c-FLIP was used to infect CMECs to overexpress the c-FLIP protein; siRNA-FUNDC1 targeted to silence the FUNDC1 gene, thus reducing or inhibiting the production of FUNDC1 protein. ∗ P < 0.05 vs Ctrl group, # P < 0.05 vs LPS + Ad-NC CMECs group, @ P < 0.05 vs LPS + Ad-c-FLIP CMECs group. Abbreviations as in , , , and .

Article Snippet: The samples were then incubated overnight at 4 °C with primary antibodies, including: CD34 (Abcam, ab81289, 1:100), c-FLIP (Affinity Biosciences, DF7010, 1:100), caspase-3 (Proteintech, 66470-2-Ig, 1:100), caspase-9 (Proteintech, 66169-1-Ig, 1:100), endothelial nitric oxide synthase (eNOS) (Santa Cruz Biotechnology, Sc-376751, 1:100), endothelin 1 (ET-1) (Novus Biologicals, NB300-526, 1:100), vascular endothelial cadherin (VE-cadherin) (Santa Cruz Biotechnology, sc-9989, 1:100), t-FUNDC1 (Bioss Antibodies, Bs-13227R, 1:100), and p-JNK (Santa Cruz Biotechnology, sc-6254, 1:100).

Techniques: Flow Cytometry, Permeability, Fluorescence, Membrane

Journal: JACC: Basic to Translational Science

Article Title: c-FLIP Protects Cardiac Microcirculation in Sepsis-Induced Myocardial Dysfunction Via FUNDC1-Mediated Regulation of Mitochondrial Autophagy

doi: 10.1016/j.jacbts.2025.02.016

Figure Lengend Snippet: Apoptosis and Functional Changes of CMECs in the Late Stage of LPS-Induced Injury (A and B) Optical microscopy observations of CMEC morphology and CCK-8 assay to assess cell proliferation. (C-F) Western blot analysis of changes in the expression of proteins related to the mitochondrial apoptosis pathway. (G and H) Immunofluorescence staining for caspase-9 expression on CMECs. Red fluorescence stains the specific marker CD34 on CMEC surfaces; green fluorescence stains caspase-9; blue fluorescence stains nuclei. (I and J) Flow cytometric analysis to detect apoptosis rates. (K and L) Transwell assays to assess the chemotactic motility of endothelial cells. (M): TER assay to evaluate endothelial barrier function. Note: n = 3/group; 3 technical replicates; mean ± SEM; analysis of variance with Tukey’s post hoc test. Ad-c-FLIP was used to infect CMECs to overexpress the c-FLIP protein; siRNA-FUNDC1 targeted to silence the FUNDC1 gene, thus reducing or preventing the production of FUNDC1 protein. ∗ P < 0.05 vs Ctrl group, # P < 0.05 vs LPS + Ad-NC CMECs group, @ P < 0.05 vs LPS + Ad-c-FLIP CMECs group. Abbreviations as in , , , and .

Article Snippet: The samples were then incubated overnight at 4 °C with primary antibodies, including: CD34 (Abcam, ab81289, 1:100), c-FLIP (Affinity Biosciences, DF7010, 1:100), caspase-3 (Proteintech, 66470-2-Ig, 1:100), caspase-9 (Proteintech, 66169-1-Ig, 1:100), endothelial nitric oxide synthase (eNOS) (Santa Cruz Biotechnology, Sc-376751, 1:100), endothelin 1 (ET-1) (Novus Biologicals, NB300-526, 1:100), vascular endothelial cadherin (VE-cadherin) (Santa Cruz Biotechnology, sc-9989, 1:100), t-FUNDC1 (Bioss Antibodies, Bs-13227R, 1:100), and p-JNK (Santa Cruz Biotechnology, sc-6254, 1:100).

Techniques: Functional Assay, Microscopy, CCK-8 Assay, Western Blot, Expressing, Immunofluorescence, Staining, Fluorescence, Marker

Journal: JACC: Basic to Translational Science

Article Title: c-FLIP Protects Cardiac Microcirculation in Sepsis-Induced Myocardial Dysfunction Via FUNDC1-Mediated Regulation of Mitochondrial Autophagy

doi: 10.1016/j.jacbts.2025.02.016

Figure Lengend Snippet: In the Late Stage of LPS-Induced Injury, JNK Participates in the Regulation of FUNDC1-Mediated Mitophagy by c-FLIP and its Potential Mechanisms (A to G) FUNDC1 mRNA expression and Western blot analysis of mitochondrial autophagy-related proteins. (H and I) Immunofluorescence measures the fusion between mitochondria and lysosomes; mitochondria are stained green, lysosomes red, nuclei blue, and merged mitochondria and lysosomes orange-yellow, indicative of mitochondrial autophagy. (J-M) Co-IP using anti–p-JNK or anti–t-FUNDC1 antibodies followed by immunoblotting with anti–t-FUNDC1 or anti–p-JNK antibodies to assess the interaction between proteins, with IgG serving as a negative control. (N and O) Immunofluorescence staining for observing the colocalization of p-JNK and t-FUNDC1, utilizing confocal microscopy to depict their interactions; p-JNK is stained green, t-FUNDC1 red, nuclei blue, and the colocalization of p-JNK and t-FUNDC1 orange, representing their interactions. Note: n = 3/group; 3 technical replicates (Co-IP done twice); mean ± SEM; analysis of variance with Tukey’s post hoc test. Ad-c-FLIP was used to infect CMECs to overexpress the c-FLIP protein; SP600125 (SP) is a JNK inhibitor, and Ani is a JNK activator. ∗ P < 0.05 vs control group, # P < 0.05 vs LPS + Ad-NC CMECs group, @ P < 0.05 vs LPS + Ad-c-FLIP CMECs group. Abbreviations as in , , , and .

Article Snippet: The samples were then incubated overnight at 4 °C with primary antibodies, including: CD34 (Abcam, ab81289, 1:100), c-FLIP (Affinity Biosciences, DF7010, 1:100), caspase-3 (Proteintech, 66470-2-Ig, 1:100), caspase-9 (Proteintech, 66169-1-Ig, 1:100), endothelial nitric oxide synthase (eNOS) (Santa Cruz Biotechnology, Sc-376751, 1:100), endothelin 1 (ET-1) (Novus Biologicals, NB300-526, 1:100), vascular endothelial cadherin (VE-cadherin) (Santa Cruz Biotechnology, sc-9989, 1:100), t-FUNDC1 (Bioss Antibodies, Bs-13227R, 1:100), and p-JNK (Santa Cruz Biotechnology, sc-6254, 1:100).

Techniques: Expressing, Western Blot, Immunofluorescence, Staining, Co-Immunoprecipitation Assay, Negative Control, Confocal Microscopy, Control

Journal: JACC: Basic to Translational Science

Article Title: c-FLIP Protects Cardiac Microcirculation in Sepsis-Induced Myocardial Dysfunction Via FUNDC1-Mediated Regulation of Mitochondrial Autophagy

doi: 10.1016/j.jacbts.2025.02.016

Figure Lengend Snippet: Protective Mechanism of c-FLIP in SIMD c-FLIP maintains myocardial microcirculation homeostasis by differentially regulating mitochondrial autophagy, whose aberration can lead to SIMD. Specifically, in the early stages, c-FLIP relies on FUNDC1 to modulate mitochondrial autophagy, with a deficiency in c-FLIP leading to enhanced mitochondrial autophagic activity. In the later stages, c-FLIP in the myocardial microcirculation regulates FUNDC1-mediated mitochondrial autophagy via the JNK pathway; a deficiency in c-FLIP results in abnormalities in this signaling pathway, causing suppressed mitochondrial autophagy activity. Changes in mitochondrial autophagy at any stage disrupt mitochondrial homeostasis, impair myocardial microcirculation, and ultimately trigger sepsis-induced myocardial dysfunction. Abbreviations as in and .

Article Snippet: The samples were then incubated overnight at 4 °C with primary antibodies, including: CD34 (Abcam, ab81289, 1:100), c-FLIP (Affinity Biosciences, DF7010, 1:100), caspase-3 (Proteintech, 66470-2-Ig, 1:100), caspase-9 (Proteintech, 66169-1-Ig, 1:100), endothelial nitric oxide synthase (eNOS) (Santa Cruz Biotechnology, Sc-376751, 1:100), endothelin 1 (ET-1) (Novus Biologicals, NB300-526, 1:100), vascular endothelial cadherin (VE-cadherin) (Santa Cruz Biotechnology, sc-9989, 1:100), t-FUNDC1 (Bioss Antibodies, Bs-13227R, 1:100), and p-JNK (Santa Cruz Biotechnology, sc-6254, 1:100).

Techniques: Activity Assay

Journal: Frontiers in Pharmacology

Article Title: AP39 alleviates HHCY-induced myocardial remodeling by regulating FUNDC1-mediated mitochondrial dynamics via S-sulfhydration of NEDD8/CUL4B

doi: 10.3389/fphar.2026.1729145

Figure Lengend Snippet: (A) GO analysis revealed that cardiomyocytes under HHCY conditions were mainly enriched in mitochondrial processes, mitochondrial function, and inner mitochondrial membrane signaling pathways. (B) GSEA of AP39-intervened samples that showed significant enrichment and upregulation of the mitophagy pathway. (C) Cystoscope analysis the top 5 ranked genes were SQSTM1, PINK1, HIF1α, FUNDC1, and TBK1. Expression of FUNDC1, LC3A/B in SD rats induced by HHCY (D) and H9c2 cardiomyocytes (E) in each group. (*p < 0.05 vs. Control; **p < 0.01 vs. Control; #p < 0.05 vs. HHCY; ##p < 0.01 vs. HHCY; $p < 0.05 vs. HHCY + AP39, $$$p < 0.005 vs. HHCY + AP39); (F) Expression of P53,P16 in H9c2 cardiomyocytes in each group after siRNA interference. (*p < 0.05 vs. Control; #p < 0.05 vs. HHCY; $$$p < 0.005 vs. HHCY + AP39); (G) Observation of changes in Mitophagy formation in each group under transmission electron microscopy. Expression of MFN1, MFN2, Drp1 in SD rats induced by HHCY (H) and H9c2 cardiomyocytes (I) in each group. (*p < 0.05 vs. Control; **p < 0.01 vs. Control; ***p < 0.005 vs. Control; #p < 0.05 vs. HHCY; ##p < 0.01 vs. HHCY; ###p < 0.005 vs. HHCY; $p < 0.05 vs. HHCY + AP39, $$p < 0.01 vs. HHCY + AP39, $$$p < 0.005 vs. HHCY + AP39); (J) Co-localization of FUNDC1 and LC3B in cardiomyocytes in each group under fluorescence microscopy. (K) Mitochondrial membrane potential changes after siRNA interference. (Red fluorescence indicates high mitochondrial membrane potential; green fluorescence indicates low mitochondrial membrane potential). Gene Ontology: GO; high homocysteine: HHCY; Gene Set Enrichment Analysis: GSEA.

Article Snippet: All primary antibodies used for Western blotting (WB) and immunofluorescence (IF) were purchased from the corresponding commercial suppliers, and their detailed information is listed as follows: GAPDH (Proteintech, USA; 10494-1-AP; WB 1:8000), α-smooth muscle actin (α-SMA) (Proteintech, USA; 14395-1-AP; WB 1:4000), Collagen type III (Proteintech, USA; 22734-1-AP; WB 1:800), FUNDC1 (Proteintech, China; AWA51295 ; IF 1:100, WB 1:1000), LC3B (Proteintech, USA; 14600-1-AP; IF 1:500, WB 1:4000), MFN1 (Proteintech, USA; 13798-1-AP; WB 1:5000), MFN2 (Proteintech, USA; 12186-1-AP; WB 1:20000), DRP1 (Proteintech, USA; 12957-1-AP; WB 1:5000), P53 (Proteintech, USA; 10442-1-AP; WB 1:20000), P16 (Proteintech, USA; 10883-1-AP; WB 1:3000), NEDD8 (Abways, China; AWA11003 ; WB 1:1000), and CUL4B (Abways, China; AWA48229 ; WB 1:1000).

Techniques: Membrane, Protein-Protein interactions, Expressing, Control, Transmission Assay, Electron Microscopy, Fluorescence, Microscopy

Journal: Frontiers in Pharmacology

Article Title: AP39 alleviates HHCY-induced myocardial remodeling by regulating FUNDC1-mediated mitochondrial dynamics via S-sulfhydration of NEDD8/CUL4B

doi: 10.3389/fphar.2026.1729145

Figure Lengend Snippet: (A) Co-Immunoprecipitation (Co-IP) analysis showing the binding of FUNDC1 to DRP1 in cardiomyocytes of each group; (B) Expression of P53, P16 in H9c2 cardiomyocytes in each group after siRNA interference. (*p < 0.05 vs. Control; **p < 0.01 vs. Control; #p < 0.05 vs. HHCY; ##p < 0.01 vs. HHCY; $p < 0.05 vs. HHCY + AP39); (C) Detection of cellular senescence in heart tissues of each group (upper panel) and in cardiomyocytes of each group (lower panel) by β-galactosidase assay.

Article Snippet: All primary antibodies used for Western blotting (WB) and immunofluorescence (IF) were purchased from the corresponding commercial suppliers, and their detailed information is listed as follows: GAPDH (Proteintech, USA; 10494-1-AP; WB 1:8000), α-smooth muscle actin (α-SMA) (Proteintech, USA; 14395-1-AP; WB 1:4000), Collagen type III (Proteintech, USA; 22734-1-AP; WB 1:800), FUNDC1 (Proteintech, China; AWA51295 ; IF 1:100, WB 1:1000), LC3B (Proteintech, USA; 14600-1-AP; IF 1:500, WB 1:4000), MFN1 (Proteintech, USA; 13798-1-AP; WB 1:5000), MFN2 (Proteintech, USA; 12186-1-AP; WB 1:20000), DRP1 (Proteintech, USA; 12957-1-AP; WB 1:5000), P53 (Proteintech, USA; 10442-1-AP; WB 1:20000), P16 (Proteintech, USA; 10883-1-AP; WB 1:3000), NEDD8 (Abways, China; AWA11003 ; WB 1:1000), and CUL4B (Abways, China; AWA48229 ; WB 1:1000).

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Binding Assay, Expressing, Control

Journal: Frontiers in Pharmacology

Article Title: AP39 alleviates HHCY-induced myocardial remodeling by regulating FUNDC1-mediated mitochondrial dynamics via S-sulfhydration of NEDD8/CUL4B

doi: 10.3389/fphar.2026.1729145

Figure Lengend Snippet: (A) KEGG analysis revealed that cardiomyocytes under HHCY conditions were mainly enriched in ubiquitination pathway. (B) Heatmap showing differentially expressed CULLIN family genes in HHCY hearts. P value is shown. (C) Expression of NEDD8, Cul4b in SD rats induced by HHCY in each group. (*p < 0.05 vs. Control; #p < 0.05 vs. HHCY); (D) Co-Immunoprecipitation (Co-IP) analysis showing the binding of FUNDC1 to Cul4b in cardiomyocytes of each group; (E–G) Expression of Cul4b, FUNDC1, P53, P16 in H9c2 cardiomyocytes in each group. (*p < 0.05 vs. Control; #p < 0.05 vs. HHCY; ##p < 0.01 vs. HHCY); (H) Co-immunoprecipitation (Co - IP) and ubiquitination assays were performed to assess FUNDC1 ubiquitination in Control, HHCY, and HHCY + AP39 groups. (I,J) Expression of FUNDC1, NEDD8, CUL48in H9c2 cardiomyocytes in each group. (K) Expression of NEDD8, CUL48, S-NEDD8, S-CUL4B in H9c2 cardiomyocytes in each group. (*p < 0.05 vs. Control; #p < 0.05 vs. HHCY; $p < 0.05 vs. HHCY + AP39).

Article Snippet: All primary antibodies used for Western blotting (WB) and immunofluorescence (IF) were purchased from the corresponding commercial suppliers, and their detailed information is listed as follows: GAPDH (Proteintech, USA; 10494-1-AP; WB 1:8000), α-smooth muscle actin (α-SMA) (Proteintech, USA; 14395-1-AP; WB 1:4000), Collagen type III (Proteintech, USA; 22734-1-AP; WB 1:800), FUNDC1 (Proteintech, China; AWA51295 ; IF 1:100, WB 1:1000), LC3B (Proteintech, USA; 14600-1-AP; IF 1:500, WB 1:4000), MFN1 (Proteintech, USA; 13798-1-AP; WB 1:5000), MFN2 (Proteintech, USA; 12186-1-AP; WB 1:20000), DRP1 (Proteintech, USA; 12957-1-AP; WB 1:5000), P53 (Proteintech, USA; 10442-1-AP; WB 1:20000), P16 (Proteintech, USA; 10883-1-AP; WB 1:3000), NEDD8 (Abways, China; AWA11003 ; WB 1:1000), and CUL4B (Abways, China; AWA48229 ; WB 1:1000).

Techniques: Ubiquitin Proteomics, Expressing, Control, Immunoprecipitation, Co-Immunoprecipitation Assay, Binding Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Fibroblast growth factor-inducible 14 accelerates pulmonary fibrosis by inducing fibroblast senescence in mice

doi: 10.1007/s00018-026-06161-w

Figure Lengend Snippet: Activation of Fn14 inhibits mitophagy in fibroblasts. A , primary fibroblasts were treated with rTWEAK (100 ng/mL) for 48 h. Cell samples were harvested after 48 h. Volcano plot showing the changes of fibroblast genes (fold change ≥ 2). B , KEGG enrichment analysis for differentially expressed genes in Control vs. rTWEAK-treated primary fibroblasts. C , Mitochondrial membrane potential was measured by JC-1 staining. D , the expression of Mfn1 , Opa1 , and Dnm1 mRNA in primary fibroblasts was detected by Real-time PCR ( n = 3). E-J , The protein levels of L-OPA1, MFN1, FUNDC1, PINK1, and LC3 were determined by Western blot analysis ( n = 3). K , Immunofluorescence and confocal microscopy show the colocalization of Mito Tracker (green) and LAMP1 (red) in rTWEAK-treated primary fibroblasts, scale bar = 50 μm. * P < 0.05 and ** P < 0.01

Article Snippet: Anti-FUNDC1 monoclonal antibody , CST , 49,240 , 1: 2000.

Techniques: Activation Assay, Control, Membrane, Staining, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Immunofluorescence, Confocal Microscopy

Journal: Nature Communications

Article Title: Endothelial FUNDC1 regulates metabolic reprogramming and the obesity-diabetes transition through the SIRT3/GATA2/endothelin-1 axis

doi: 10.1038/s41467-026-68548-4

Figure Lengend Snippet: a – c CD31 positive ECs (CD31 + ECs) were isolated from white adipose tissue (WAT) of mice fed with indicated durations (2, 4, 6 months) of HFD and subjected to western blot analysis ( n = 4 mice/group) or Q-PCR analysis ( n = 6 mice/group). d – f HUVECs were incubated with 200 μM PA conjugated to fatty acid-free BSA, and culture medium containing PA or BSA was refreshed daily throughout the 7-day stimulation period to maintain a consistent lipid challenge. Cells were then subjected to western blot analysis or Q-PCR analysis. n = 3 independent experiments. g CD31 positive cells were removed from the SVF of Fundc1 f/Y Cdh5 - or Fundc1 f/Y Cdh5 + mice (CD31 - SVF). Normal SVF containing CD31 positive cells was used as control (CD31 + SVF). Cells were then differentiated into adipocytes. Scale bar: 50 µm. h Intracellular TG content in ( g ). n = 6 independent experiments. i Insulin-stimulated 2-NBDG uptake in primary adipocytes isolated from Fundc1 f/Y Cdh5 - or Fundc1 f/Y Cdh5 + mice in the present of bovine serum albumin (BSA) or palmitic acid (PA). n = 8 independent experiments. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, n.s. p > 0.05.

Article Snippet: Immunofluorescence staining in HUVECs were performed by incubating the methanol-fixed cells with primary antibodies against FUNDC1 (Biorbyt, orb156964), SIRT3 (Santa Cruz Biotechnology, sc-365175), GATA2 (Proteintech, 11103-1-AP), or isotype control at 4 °C overnight, and followed by incubation with corresponding fluorescent secondary antibodies (Life Technologies) at room temperature for 1 h. Nuclei were stained with 4’6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, D9542).

Techniques: Isolation, Western Blot, Incubation, Control

Journal: Nature Communications

Article Title: Endothelial FUNDC1 regulates metabolic reprogramming and the obesity-diabetes transition through the SIRT3/GATA2/endothelin-1 axis

doi: 10.1038/s41467-026-68548-4

Figure Lengend Snippet: a – l Fundc1 f/Y Cdh5 + mice and littermates ( Fundc1 f/Y Cdh5 - ) were kept on HFD for 6 months. a Changes in body weight during HFD feeding. * p < 0.05, ** p < 0.01 (HFD- Fundc1 f/Y Cdh5 - vs . HFD- Fundc1 f/Y Cdh5 +) , n = 15–20. b Fat percentage of mice. n = 15–20 mice/group. c Adipose tissue weight/body weight. n = 15–20 mice/group. d Representative images of H&E staining of SubWAT. e Quantification of siz e s and numbers of adipocytes under HFD feeding. n = 15–20 mice/group. f Quantification of adipocyte diameter. n = 15–20 mice/group. g Glucose tolerance test (GTT) and quantification of the area under curve, n = 7-8 mice/group. h Insulin tolerance test (ITT) and quantification of the area under curve, n = 7–8 mice/group. i , j Whole body respiratory exchange ratio (RER; VCO 2 /VO 2 , n = 8 mice/group). k , l Energy expenditure normalized to body weight, n = 8 mice/group. Data are presented as mea n ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, n.s. p > 0.05.

Article Snippet: Immunofluorescence staining in HUVECs were performed by incubating the methanol-fixed cells with primary antibodies against FUNDC1 (Biorbyt, orb156964), SIRT3 (Santa Cruz Biotechnology, sc-365175), GATA2 (Proteintech, 11103-1-AP), or isotype control at 4 °C overnight, and followed by incubation with corresponding fluorescent secondary antibodies (Life Technologies) at room temperature for 1 h. Nuclei were stained with 4’6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, D9542).

Techniques: Staining

Journal: Nature Communications

Article Title: Endothelial FUNDC1 regulates metabolic reprogramming and the obesity-diabetes transition through the SIRT3/GATA2/endothelin-1 axis

doi: 10.1038/s41467-026-68548-4

Figure Lengend Snippet: Mice were placed on a high-fat diet (HFD) and infused with ET-1 (10 µg/kg/day) or saline concurrently, starting at the onset of HFD feeding, using subcutaneously implanted osmotic pumps for 4 weeks. a Representative pictures of whole body, epididymal white adipose tissue (EpiWAT), subcutaneous white adipose tissue (SubWAT), and brown adipose tissue (BAT). b Changes in body weight during HFD feeding periods (left panel), * p < 0.05, ** p < 0.01 (Saline- Fundc1 f/Y Cdh5 - vs . Saline- Fundc1 f/Y Cdh5 + ), # p < 0.05, ## p < 0.01 (ET-1- Fundc1 f/Y Cdh5 - vs . ET-1- Fundc1 f/Y Cdh5 + ), and relative weights of adipose tissues ( right panel). n = 7–8 mice/group. c Glucose tolerance test (IPGTT) and insulin tolerance test (ITT). n = 7–8 mice/group. d Fasting blood glucose (GLU) level. n = 7–8 mice/group. e Serum insulin level. n = 7–8 mice/group. f Serum free fatty acid (FFA) level. n = 7–8 mice/group. g – i Aortic ring vasoreactivity assay in ET-1-infused mice. n = 6 mice/group. j , k Circumferential cyclic strain ( j ) and (PWV) pulse wave velocity ( k ) of carotid artery. n = 6 mice/group. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, n.s. p > 0.05.

Article Snippet: Immunofluorescence staining in HUVECs were performed by incubating the methanol-fixed cells with primary antibodies against FUNDC1 (Biorbyt, orb156964), SIRT3 (Santa Cruz Biotechnology, sc-365175), GATA2 (Proteintech, 11103-1-AP), or isotype control at 4 °C overnight, and followed by incubation with corresponding fluorescent secondary antibodies (Life Technologies) at room temperature for 1 h. Nuclei were stained with 4’6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, D9542).

Techniques: Saline

Journal: Nature Communications

Article Title: Endothelial FUNDC1 regulates metabolic reprogramming and the obesity-diabetes transition through the SIRT3/GATA2/endothelin-1 axis

doi: 10.1038/s41467-026-68548-4

Figure Lengend Snippet: a HUVECs were transfected with control siRNA (siCtrl) or FUNDC1 siRNA ( siFUNDC1 ) and subjected to immunoblotting analysis. b Quantification of protein levels of ECE1, GATA2, and AP1. n = 6 independent experiments. c HUVECs were transfected with control siRNA (siCtrl) or FUNDC1 siRNA ( siFUNDC1 ) and subjected to Q-PCR analysis to detect ECE1, AP1, and JUN mRNA levels. n = 8 independent experiments. d HUVECs were transfected with control Myc-Flag-tagged plasmid ( Myc-Flag-Ctrl ) or Myc-Flag-FUNDC1 plasmid ( Myc-Flag-FUNDC1 ) and subjected to immunoblotting analysis. e Quantification of protein levels of FUNDC1, ECE1, GATA2 and AP1 in ( d ). n = 6 independent experiments. f HUVECs were transfected with control siRNA (siCtrl) or GATA2 siRNA ( siGATA2 ) and subjected to Q-PCR analysis to detect EDN1 mRNA levels. n = 8 independent experiments. g HUVECs were transfected with control siRNA (siCtrl) or FUNDC1 siRNA ( siFUNDC1 ) and were then treat with 200 µM PA for indicated time periods. h - j Quantification of protein levels of FUNDC1, GATA2, and ET-1 in ( g ). n = 4 independent experiments. Data are presented as mean ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001, n.s. p > 0.05.

Article Snippet: Immunofluorescence staining in HUVECs were performed by incubating the methanol-fixed cells with primary antibodies against FUNDC1 (Biorbyt, orb156964), SIRT3 (Santa Cruz Biotechnology, sc-365175), GATA2 (Proteintech, 11103-1-AP), or isotype control at 4 °C overnight, and followed by incubation with corresponding fluorescent secondary antibodies (Life Technologies) at room temperature for 1 h. Nuclei were stained with 4’6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, D9542).

Techniques: Transfection, Control, Western Blot, Plasmid Preparation

Journal: Nature Communications

Article Title: Endothelial FUNDC1 regulates metabolic reprogramming and the obesity-diabetes transition through the SIRT3/GATA2/endothelin-1 axis

doi: 10.1038/s41467-026-68548-4

Figure Lengend Snippet: a HUVECs were transfected with control siRNA (siCtrl) or SIRT3 siRNA ( siSIRT3 ) and subjected to immunoblotting analysis. b Quantification of protein levels in ( a ). n = 8–10 independent experiments. c HUVECs were transfected with control siRNA (siCtrl) or SIRT3 siRNA ( siSIRT3 ) and subjected to Q-PCR analysis to detect mRNA levels of GATA2 and EDN1 . n = 8 independent experiments. d The interactions between SIRT3 and FUNDC1 or GATA2 in HUVECs were determined by immunoprecipitation (IP) and immunoblotting (IB) analysis. e The co-localization of SIRT3 and FUNDC1 or GATA2 were determined by immunofluorescence staining. Nuclei were stained by DAPI. MERGE1 indicates merge of SIRT3 and DAPI; MERGE2 indicates merge of FUNDC1, SIRT3, and DAPI; MERGE3 indicates merge of GATA2 and DAPI; MERGE4 indicates merge of GATA2, SIRT3, and DAPI. Scale bar: 10 µm. f HUVECs were transfected with control siRNA (siCtrl) or FUNDC1 siRNA ( siFUNDC1 ) in the presence of BSA or 200 µM PA and subjected to immunofluorescence staining analysis. MERGE1 indicates merge of SIRT3 and DAPI; MERGE2 indicates merge of FUNDC1, SIRT3, and DAPI. Scale bar: 10 µm. g Pearson’s coefficient calculated by ZEN software, indicates the co-localization of FUNDC1 and SIRT3. n = 9 cells from three independent experiments. h Quantification of nuclear SIRT3 level. i HUVECs were transfected with control siRNA (siCtrl) or FUNDC1 siRNA ( siFUN ) in the presence of BSA or 200 µM PA. Non-nuclear fraction (Non-Nuc), nuclear fraction (Nuc), and mitochondria fraction (Mito) were prepared and subjected to immunoblotting analysis. n = 9 cells from three independent experiments. j Quantification of protein level of SIRT3 long isoform (SIRT3-L) in the nucleus (Nuclear SIRT3-L) and in mitochondria (Mitochondrial SIRT3-L). n = 4 independent experiments. k HUVECs were double transfected with control siRNA (siCtrl) or FUNDC1 ( siFUN ) and SIRT3 siRNA and subjected to immunoblotting analysis. l Quantification of GATA2 protein level in ( k ). n = 5 independent experiments. m HUVECs were double transfected with either control siRNA (siCtrl) or FUNDC1 and SIRT3 siRNA and subjected to Q-PCR analysis to detect END1 mRNA level. n = 8 independent experiments. Data are presented as mean ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001, n.s. p > 0.05.

Article Snippet: Immunofluorescence staining in HUVECs were performed by incubating the methanol-fixed cells with primary antibodies against FUNDC1 (Biorbyt, orb156964), SIRT3 (Santa Cruz Biotechnology, sc-365175), GATA2 (Proteintech, 11103-1-AP), or isotype control at 4 °C overnight, and followed by incubation with corresponding fluorescent secondary antibodies (Life Technologies) at room temperature for 1 h. Nuclei were stained with 4’6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, D9542).

Techniques: Transfection, Control, Western Blot, Immunoprecipitation, Immunofluorescence, Staining, Software

Journal: Nature Communications

Article Title: Endothelial FUNDC1 regulates metabolic reprogramming and the obesity-diabetes transition through the SIRT3/GATA2/endothelin-1 axis

doi: 10.1038/s41467-026-68548-4

Figure Lengend Snippet: a HUVECs were transfected with control siRNA (siCtrl) or SIRT3 siRNA and then treated with 50 µg/ml cycloheximide (CHX) for 240 min. Cell lysates were subjected to immunoblotting analysis. b Quantification of GATA2 protein level in ( a ). n = 4 independent experiments. c Acetylated GATA2 level was determined by IP of acetylate lysine (Ac-lysine) in cells transfected with SIRT3 siRNA and followed by immunoblotting (IB) of GATA2. d Quantification of the enrichment of acetylated GATA2 in ( c ). n = 4 independent experiments. e Acetylated GATA2 level was determined by IP of Ac-lysine in cells treated with PA (200 µM) and followed by immunoblotting (IB) GATA2. f Quantification of the enrichment of acetylated GATA2 in ( e ). n = 4 independent experiments. g HUVECs were transfected with control siRNA (siCtrl) or SIRT3 siRNA ( siSIRT3 ) and subjected to immunoblotting analysis. h HUVECs were transfected with control Myc-Flag-tagged plasmid ( Myc-Flag-Ctrl ) or Myc-Flag-SIRT3 plasmid ( Myc-Flag-SIRT3 ) and subjected to immunoblotting analysis. i , j Quantification of protein levels of FUNDC1. n = 6–9 independent experiments. k HUVECs were transfected with control siRNA (siCtrl) or SIRT3 siRNA ( siSIRT3 and subjected to Q-PCR analysis to detect FUNDC1 mRNA level. n = 8. l HUVECs were transfected with control siRNA (siCtrl) or SIRT3 siRNA and then treated with 50 µg/ml cycloheximide (CHX) for 240 min. Cell lysates were subjected to immunoblotting analysis. m Quantification of GATA2 protein level in ( l ). n = 3 independent experiments. Data are presented as mean ± SD, in ( b and m ), star represents the comparison between groups siCtrl and siSIRT3 at the same treatment intervals, pound represents the comparison with control group (time = 0 min). * p < 0.05, ** p < 0.01, *** p < 0.001, n.s. p > 0.05. # p < 0.05, ## p < 0.01, ### p < 0.001, n.s. p > 0.05.

Article Snippet: Immunofluorescence staining in HUVECs were performed by incubating the methanol-fixed cells with primary antibodies against FUNDC1 (Biorbyt, orb156964), SIRT3 (Santa Cruz Biotechnology, sc-365175), GATA2 (Proteintech, 11103-1-AP), or isotype control at 4 °C overnight, and followed by incubation with corresponding fluorescent secondary antibodies (Life Technologies) at room temperature for 1 h. Nuclei were stained with 4’6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, D9542).

Techniques: Transfection, Control, Western Blot, Plasmid Preparation, Comparison

Journal: Nature Communications

Article Title: Endothelial FUNDC1 regulates metabolic reprogramming and the obesity-diabetes transition through the SIRT3/GATA2/endothelin-1 axis

doi: 10.1038/s41467-026-68548-4

Figure Lengend Snippet: a Potential KFERQ-like motifs of human FUNDC1 and SIRT3. b Interactions between HSC70 with FUNDC1 and SIRT3-L in HUVECs were detected by IP and IB. c Co-staining of FUNDC1 with HSC70 and SIRT3-L by IF. d Interaction of SIRT3-L and FUNDC1 was analyzed in HUVECs transfected with HSC70 siRNA. e Quantification of protein levels in HUVECS transfected with HSC70 siRNA. f Quantification of the binding activity of FUNDC1 with SIRT3-L in HSC70 knockdown cells, indicated by the enrichment of FUNDC1. n = 4 independent experiments. g HUVECs were transfected with control siRNA (siCtrl) or HSC70 siRNA ( siHSC70 ) in the presence of BSA or 200 µM PA. Non-nuclear fraction (Non-Nuc), nuclear fraction (Nuc), and mitochondria fraction (Mito) were prepared and subjected to immunoblotting analysis. h Quantification of protein level of SIRT3 long isoform (SIRT3-L) in the nucleus (Nuclear SIRT3-L) and in mitochondria (Mitochondrial SIRT3-L). n = 4 independent experiments. Data are presented as mean ± SD, * p < 0.05, ** p < 0.01 derived from Student’s t tests.

Article Snippet: Immunofluorescence staining in HUVECs were performed by incubating the methanol-fixed cells with primary antibodies against FUNDC1 (Biorbyt, orb156964), SIRT3 (Santa Cruz Biotechnology, sc-365175), GATA2 (Proteintech, 11103-1-AP), or isotype control at 4 °C overnight, and followed by incubation with corresponding fluorescent secondary antibodies (Life Technologies) at room temperature for 1 h. Nuclei were stained with 4’6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, D9542).

Techniques: Staining, Transfection, Binding Assay, Activity Assay, Knockdown, Control, Western Blot, Derivative Assay

Journal: Nature Communications

Article Title: Endothelial FUNDC1 regulates metabolic reprogramming and the obesity-diabetes transition through the SIRT3/GATA2/endothelin-1 axis

doi: 10.1038/s41467-026-68548-4

Figure Lengend Snippet: Plasma and endothelium were collected from age-matched healthy donors ( n = 17) or donors with both obesity and T2DM (Obesity&T2DM, n = 19). a Characteristics of human donors. Age, body mass index (BMI), and glycohemoglobin A1c (HbA1c) levels are represented as mean ± SD. b Plasma ET-1 level in human donors. n = 17–19 donors/group. c Spearman’s correlation of plasma ET-1 level and BMI. d Spearman’s correlation of plasma ET-1 level and HbA1c. e qPCR analysis of FUNDC1 , END1 and GATA2 mRNA levels in the intima isolated from human small arterioles. n = 17–19 donors/group. f Spearman’s correlation of mRNA levels of FUNDC1 and END1 . g Spearman’s correlation of mRNA levels of GATA2 and END1 . h Spearman’s correlation of intimal FUNDC1 mRNA and plasma ET-1. i , j Human small arterioles were collected from age-matched healthy donors or donors with both obesity and T2DM (Obesity&T2DM) and subjected to western blot analysis ( i and j ). n = 6 donors/group. k , l Human femoral arteries were collected from age-matched healthy donors (n = 7) or donors with both obesity and T2DM (Obesity&T2DM; n = 5) and subjected to IHC staining. Scale bar: 50 µm. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, n.s. p > 0.05.

Article Snippet: Immunofluorescence staining in HUVECs were performed by incubating the methanol-fixed cells with primary antibodies against FUNDC1 (Biorbyt, orb156964), SIRT3 (Santa Cruz Biotechnology, sc-365175), GATA2 (Proteintech, 11103-1-AP), or isotype control at 4 °C overnight, and followed by incubation with corresponding fluorescent secondary antibodies (Life Technologies) at room temperature for 1 h. Nuclei were stained with 4’6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, D9542).

Techniques: Clinical Proteomics, Isolation, Western Blot, Immunohistochemistry

Journal: Nature Communications

Article Title: Endothelial FUNDC1 regulates metabolic reprogramming and the obesity-diabetes transition through the SIRT3/GATA2/endothelin-1 axis

doi: 10.1038/s41467-026-68548-4

Figure Lengend Snippet: In the healthy state, endothelial FUNDC1 anchors SIRT3-L in mitochondria, limiting its nuclear accumulation and regulating its interaction with GATA2, thereby maintaining normal ET-1 production and preserving vascular and metabolic homeostasis. Under overnutrition-induced metabolic stress, SIRT3-L is released from GATA2, contributing to enhanced GATA2-mediated ET-1 transcription. Elevated ET-1 promote angiogenesis to provide the space for adipocyte hyperplasia at the early stage and later exacerbate insulin resistance and thus driving the progression from obesity to T2DM. Importantly, SIRT3-L translocation to mitochondria also facilitates FUNDC1 degradation at the early obese stage, which triggers a compensatory increase in FUNDC1 transcription and ultimately results in FUNDC1 overexpression in a late-stage. The elevation of FUNDC1 further enhances mitochondrial recruitment of SIRT3-L, amplifying ET-1 production. In contrast, in Fundc1 EC-specific knockout (ECKO) mice, the absence of FUNDC1 prevents mitochondrial recruitment of SIRT3-L, leading to its nuclear retention, where SIRT3-L promotes GATA2 degradation and suppresses ET-1 transcription. The loss of Fundc1 in ECs not only suppresses angiogenesis at early overnutrition stage but also prevents EC senescence at late stage to attenuate the potential diabetic vascular complications.

Article Snippet: Immunofluorescence staining in HUVECs were performed by incubating the methanol-fixed cells with primary antibodies against FUNDC1 (Biorbyt, orb156964), SIRT3 (Santa Cruz Biotechnology, sc-365175), GATA2 (Proteintech, 11103-1-AP), or isotype control at 4 °C overnight, and followed by incubation with corresponding fluorescent secondary antibodies (Life Technologies) at room temperature for 1 h. Nuclei were stained with 4’6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, D9542).

Techniques: Preserving, Translocation Assay, Over Expression, Knock-Out

Journal: Nature Communications

Article Title: Endothelial FUNDC1 regulates metabolic reprogramming and the obesity-diabetes transition through the SIRT3/GATA2/endothelin-1 axis

doi: 10.1038/s41467-026-68548-4

Figure Lengend Snippet: a – c CD31 positive ECs (CD31 + ECs) were isolated from white adipose tissue (WAT) of mice fed with indicated durations (2, 4, 6 months) of HFD and subjected to western blot analysis ( n = 4 mice/group) or Q-PCR analysis ( n = 6 mice/group). d – f HUVECs were incubated with 200 μM PA conjugated to fatty acid-free BSA, and culture medium containing PA or BSA was refreshed daily throughout the 7-day stimulation period to maintain a consistent lipid challenge. Cells were then subjected to western blot analysis or Q-PCR analysis. n = 3 independent experiments. g CD31 positive cells were removed from the SVF of Fundc1 f/Y Cdh5 - or Fundc1 f/Y Cdh5 + mice (CD31 - SVF). Normal SVF containing CD31 positive cells was used as control (CD31 + SVF). Cells were then differentiated into adipocytes. Scale bar: 50 µm. h Intracellular TG content in ( g ). n = 6 independent experiments. i Insulin-stimulated 2-NBDG uptake in primary adipocytes isolated from Fundc1 f/Y Cdh5 - or Fundc1 f/Y Cdh5 + mice in the present of bovine serum albumin (BSA) or palmitic acid (PA). n = 8 independent experiments. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, n.s. p > 0.05.

Article Snippet: Membranes were probed with primary antibodies against FUNDC1 (Novus, NBP1-81063), ECE1 (Santa Cruz Biotechnology, sc-376018), GATA2 (Novus, NBP1-82581; Santa Cruz Biotechnology, sc-267), AP-1 (Cell Signaling Technology, 9165), SIRT3-L (LSBio, LS- C46967 ), SIRT3-S (Cell Signaling Technology, 5490), COX IV (Cell Signaling Technology, 4850), Lamin A/C (Cell Signaling Technology, 4777), ET-1 (Santa Cruz Biotechnology, sc-517436), p-AKT Ser473 (Cell Signaling Technology, 4060), AKT (Cell Signaling Technology, 4685), β-actin (Santa Cruz Biotechnology, 47778), or GAPDH (Santa Cruz Biotechnology, 32233), and subsequently incubated with horseradish peroxidase-linked secondary antibodies.

Techniques: Isolation, Western Blot, Incubation, Control

Journal: Nature Communications

Article Title: Endothelial FUNDC1 regulates metabolic reprogramming and the obesity-diabetes transition through the SIRT3/GATA2/endothelin-1 axis

doi: 10.1038/s41467-026-68548-4

Figure Lengend Snippet: a – l Fundc1 f/Y Cdh5 + mice and littermates ( Fundc1 f/Y Cdh5 - ) were kept on HFD for 6 months. a Changes in body weight during HFD feeding. * p < 0.05, ** p < 0.01 (HFD- Fundc1 f/Y Cdh5 - vs . HFD- Fundc1 f/Y Cdh5 +) , n = 15–20. b Fat percentage of mice. n = 15–20 mice/group. c Adipose tissue weight/body weight. n = 15–20 mice/group. d Representative images of H&E staining of SubWAT. e Quantification of siz e s and numbers of adipocytes under HFD feeding. n = 15–20 mice/group. f Quantification of adipocyte diameter. n = 15–20 mice/group. g Glucose tolerance test (GTT) and quantification of the area under curve, n = 7-8 mice/group. h Insulin tolerance test (ITT) and quantification of the area under curve, n = 7–8 mice/group. i , j Whole body respiratory exchange ratio (RER; VCO 2 /VO 2 , n = 8 mice/group). k , l Energy expenditure normalized to body weight, n = 8 mice/group. Data are presented as mea n ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, n.s. p > 0.05.

Article Snippet: Membranes were probed with primary antibodies against FUNDC1 (Novus, NBP1-81063), ECE1 (Santa Cruz Biotechnology, sc-376018), GATA2 (Novus, NBP1-82581; Santa Cruz Biotechnology, sc-267), AP-1 (Cell Signaling Technology, 9165), SIRT3-L (LSBio, LS- C46967 ), SIRT3-S (Cell Signaling Technology, 5490), COX IV (Cell Signaling Technology, 4850), Lamin A/C (Cell Signaling Technology, 4777), ET-1 (Santa Cruz Biotechnology, sc-517436), p-AKT Ser473 (Cell Signaling Technology, 4060), AKT (Cell Signaling Technology, 4685), β-actin (Santa Cruz Biotechnology, 47778), or GAPDH (Santa Cruz Biotechnology, 32233), and subsequently incubated with horseradish peroxidase-linked secondary antibodies.

Techniques: Staining

Journal: Nature Communications

Article Title: Endothelial FUNDC1 regulates metabolic reprogramming and the obesity-diabetes transition through the SIRT3/GATA2/endothelin-1 axis

doi: 10.1038/s41467-026-68548-4

Figure Lengend Snippet: Mice were placed on a high-fat diet (HFD) and infused with ET-1 (10 µg/kg/day) or saline concurrently, starting at the onset of HFD feeding, using subcutaneously implanted osmotic pumps for 4 weeks. a Representative pictures of whole body, epididymal white adipose tissue (EpiWAT), subcutaneous white adipose tissue (SubWAT), and brown adipose tissue (BAT). b Changes in body weight during HFD feeding periods (left panel), * p < 0.05, ** p < 0.01 (Saline- Fundc1 f/Y Cdh5 - vs . Saline- Fundc1 f/Y Cdh5 + ), # p < 0.05, ## p < 0.01 (ET-1- Fundc1 f/Y Cdh5 - vs . ET-1- Fundc1 f/Y Cdh5 + ), and relative weights of adipose tissues ( right panel). n = 7–8 mice/group. c Glucose tolerance test (IPGTT) and insulin tolerance test (ITT). n = 7–8 mice/group. d Fasting blood glucose (GLU) level. n = 7–8 mice/group. e Serum insulin level. n = 7–8 mice/group. f Serum free fatty acid (FFA) level. n = 7–8 mice/group. g – i Aortic ring vasoreactivity assay in ET-1-infused mice. n = 6 mice/group. j , k Circumferential cyclic strain ( j ) and (PWV) pulse wave velocity ( k ) of carotid artery. n = 6 mice/group. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, n.s. p > 0.05.

Article Snippet: Membranes were probed with primary antibodies against FUNDC1 (Novus, NBP1-81063), ECE1 (Santa Cruz Biotechnology, sc-376018), GATA2 (Novus, NBP1-82581; Santa Cruz Biotechnology, sc-267), AP-1 (Cell Signaling Technology, 9165), SIRT3-L (LSBio, LS- C46967 ), SIRT3-S (Cell Signaling Technology, 5490), COX IV (Cell Signaling Technology, 4850), Lamin A/C (Cell Signaling Technology, 4777), ET-1 (Santa Cruz Biotechnology, sc-517436), p-AKT Ser473 (Cell Signaling Technology, 4060), AKT (Cell Signaling Technology, 4685), β-actin (Santa Cruz Biotechnology, 47778), or GAPDH (Santa Cruz Biotechnology, 32233), and subsequently incubated with horseradish peroxidase-linked secondary antibodies.

Techniques: Saline

Journal: Nature Communications

Article Title: Endothelial FUNDC1 regulates metabolic reprogramming and the obesity-diabetes transition through the SIRT3/GATA2/endothelin-1 axis

doi: 10.1038/s41467-026-68548-4

Figure Lengend Snippet: a HUVECs were transfected with control siRNA (siCtrl) or FUNDC1 siRNA ( siFUNDC1 ) and subjected to immunoblotting analysis. b Quantification of protein levels of ECE1, GATA2, and AP1. n = 6 independent experiments. c HUVECs were transfected with control siRNA (siCtrl) or FUNDC1 siRNA ( siFUNDC1 ) and subjected to Q-PCR analysis to detect ECE1, AP1, and JUN mRNA levels. n = 8 independent experiments. d HUVECs were transfected with control Myc-Flag-tagged plasmid ( Myc-Flag-Ctrl ) or Myc-Flag-FUNDC1 plasmid ( Myc-Flag-FUNDC1 ) and subjected to immunoblotting analysis. e Quantification of protein levels of FUNDC1, ECE1, GATA2 and AP1 in ( d ). n = 6 independent experiments. f HUVECs were transfected with control siRNA (siCtrl) or GATA2 siRNA ( siGATA2 ) and subjected to Q-PCR analysis to detect EDN1 mRNA levels. n = 8 independent experiments. g HUVECs were transfected with control siRNA (siCtrl) or FUNDC1 siRNA ( siFUNDC1 ) and were then treat with 200 µM PA for indicated time periods. h - j Quantification of protein levels of FUNDC1, GATA2, and ET-1 in ( g ). n = 4 independent experiments. Data are presented as mean ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001, n.s. p > 0.05.

Article Snippet: Membranes were probed with primary antibodies against FUNDC1 (Novus, NBP1-81063), ECE1 (Santa Cruz Biotechnology, sc-376018), GATA2 (Novus, NBP1-82581; Santa Cruz Biotechnology, sc-267), AP-1 (Cell Signaling Technology, 9165), SIRT3-L (LSBio, LS- C46967 ), SIRT3-S (Cell Signaling Technology, 5490), COX IV (Cell Signaling Technology, 4850), Lamin A/C (Cell Signaling Technology, 4777), ET-1 (Santa Cruz Biotechnology, sc-517436), p-AKT Ser473 (Cell Signaling Technology, 4060), AKT (Cell Signaling Technology, 4685), β-actin (Santa Cruz Biotechnology, 47778), or GAPDH (Santa Cruz Biotechnology, 32233), and subsequently incubated with horseradish peroxidase-linked secondary antibodies.

Techniques: Transfection, Control, Western Blot, Plasmid Preparation

Journal: Nature Communications

Article Title: Endothelial FUNDC1 regulates metabolic reprogramming and the obesity-diabetes transition through the SIRT3/GATA2/endothelin-1 axis

doi: 10.1038/s41467-026-68548-4

Figure Lengend Snippet: a HUVECs were transfected with control siRNA (siCtrl) or SIRT3 siRNA ( siSIRT3 ) and subjected to immunoblotting analysis. b Quantification of protein levels in ( a ). n = 8–10 independent experiments. c HUVECs were transfected with control siRNA (siCtrl) or SIRT3 siRNA ( siSIRT3 ) and subjected to Q-PCR analysis to detect mRNA levels of GATA2 and EDN1 . n = 8 independent experiments. d The interactions between SIRT3 and FUNDC1 or GATA2 in HUVECs were determined by immunoprecipitation (IP) and immunoblotting (IB) analysis. e The co-localization of SIRT3 and FUNDC1 or GATA2 were determined by immunofluorescence staining. Nuclei were stained by DAPI. MERGE1 indicates merge of SIRT3 and DAPI; MERGE2 indicates merge of FUNDC1, SIRT3, and DAPI; MERGE3 indicates merge of GATA2 and DAPI; MERGE4 indicates merge of GATA2, SIRT3, and DAPI. Scale bar: 10 µm. f HUVECs were transfected with control siRNA (siCtrl) or FUNDC1 siRNA ( siFUNDC1 ) in the presence of BSA or 200 µM PA and subjected to immunofluorescence staining analysis. MERGE1 indicates merge of SIRT3 and DAPI; MERGE2 indicates merge of FUNDC1, SIRT3, and DAPI. Scale bar: 10 µm. g Pearson’s coefficient calculated by ZEN software, indicates the co-localization of FUNDC1 and SIRT3. n = 9 cells from three independent experiments. h Quantification of nuclear SIRT3 level. i HUVECs were transfected with control siRNA (siCtrl) or FUNDC1 siRNA ( siFUN ) in the presence of BSA or 200 µM PA. Non-nuclear fraction (Non-Nuc), nuclear fraction (Nuc), and mitochondria fraction (Mito) were prepared and subjected to immunoblotting analysis. n = 9 cells from three independent experiments. j Quantification of protein level of SIRT3 long isoform (SIRT3-L) in the nucleus (Nuclear SIRT3-L) and in mitochondria (Mitochondrial SIRT3-L). n = 4 independent experiments. k HUVECs were double transfected with control siRNA (siCtrl) or FUNDC1 ( siFUN ) and SIRT3 siRNA and subjected to immunoblotting analysis. l Quantification of GATA2 protein level in ( k ). n = 5 independent experiments. m HUVECs were double transfected with either control siRNA (siCtrl) or FUNDC1 and SIRT3 siRNA and subjected to Q-PCR analysis to detect END1 mRNA level. n = 8 independent experiments. Data are presented as mean ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001, n.s. p > 0.05.

Article Snippet: Membranes were probed with primary antibodies against FUNDC1 (Novus, NBP1-81063), ECE1 (Santa Cruz Biotechnology, sc-376018), GATA2 (Novus, NBP1-82581; Santa Cruz Biotechnology, sc-267), AP-1 (Cell Signaling Technology, 9165), SIRT3-L (LSBio, LS- C46967 ), SIRT3-S (Cell Signaling Technology, 5490), COX IV (Cell Signaling Technology, 4850), Lamin A/C (Cell Signaling Technology, 4777), ET-1 (Santa Cruz Biotechnology, sc-517436), p-AKT Ser473 (Cell Signaling Technology, 4060), AKT (Cell Signaling Technology, 4685), β-actin (Santa Cruz Biotechnology, 47778), or GAPDH (Santa Cruz Biotechnology, 32233), and subsequently incubated with horseradish peroxidase-linked secondary antibodies.

Techniques: Transfection, Control, Western Blot, Immunoprecipitation, Immunofluorescence, Staining, Software

Journal: Nature Communications

Article Title: Endothelial FUNDC1 regulates metabolic reprogramming and the obesity-diabetes transition through the SIRT3/GATA2/endothelin-1 axis

doi: 10.1038/s41467-026-68548-4

Figure Lengend Snippet: a HUVECs were transfected with control siRNA (siCtrl) or SIRT3 siRNA and then treated with 50 µg/ml cycloheximide (CHX) for 240 min. Cell lysates were subjected to immunoblotting analysis. b Quantification of GATA2 protein level in ( a ). n = 4 independent experiments. c Acetylated GATA2 level was determined by IP of acetylate lysine (Ac-lysine) in cells transfected with SIRT3 siRNA and followed by immunoblotting (IB) of GATA2. d Quantification of the enrichment of acetylated GATA2 in ( c ). n = 4 independent experiments. e Acetylated GATA2 level was determined by IP of Ac-lysine in cells treated with PA (200 µM) and followed by immunoblotting (IB) GATA2. f Quantification of the enrichment of acetylated GATA2 in ( e ). n = 4 independent experiments. g HUVECs were transfected with control siRNA (siCtrl) or SIRT3 siRNA ( siSIRT3 ) and subjected to immunoblotting analysis. h HUVECs were transfected with control Myc-Flag-tagged plasmid ( Myc-Flag-Ctrl ) or Myc-Flag-SIRT3 plasmid ( Myc-Flag-SIRT3 ) and subjected to immunoblotting analysis. i , j Quantification of protein levels of FUNDC1. n = 6–9 independent experiments. k HUVECs were transfected with control siRNA (siCtrl) or SIRT3 siRNA ( siSIRT3 and subjected to Q-PCR analysis to detect FUNDC1 mRNA level. n = 8. l HUVECs were transfected with control siRNA (siCtrl) or SIRT3 siRNA and then treated with 50 µg/ml cycloheximide (CHX) for 240 min. Cell lysates were subjected to immunoblotting analysis. m Quantification of GATA2 protein level in ( l ). n = 3 independent experiments. Data are presented as mean ± SD, in ( b and m ), star represents the comparison between groups siCtrl and siSIRT3 at the same treatment intervals, pound represents the comparison with control group (time = 0 min). * p < 0.05, ** p < 0.01, *** p < 0.001, n.s. p > 0.05. # p < 0.05, ## p < 0.01, ### p < 0.001, n.s. p > 0.05.

Article Snippet: Membranes were probed with primary antibodies against FUNDC1 (Novus, NBP1-81063), ECE1 (Santa Cruz Biotechnology, sc-376018), GATA2 (Novus, NBP1-82581; Santa Cruz Biotechnology, sc-267), AP-1 (Cell Signaling Technology, 9165), SIRT3-L (LSBio, LS- C46967 ), SIRT3-S (Cell Signaling Technology, 5490), COX IV (Cell Signaling Technology, 4850), Lamin A/C (Cell Signaling Technology, 4777), ET-1 (Santa Cruz Biotechnology, sc-517436), p-AKT Ser473 (Cell Signaling Technology, 4060), AKT (Cell Signaling Technology, 4685), β-actin (Santa Cruz Biotechnology, 47778), or GAPDH (Santa Cruz Biotechnology, 32233), and subsequently incubated with horseradish peroxidase-linked secondary antibodies.

Techniques: Transfection, Control, Western Blot, Plasmid Preparation, Comparison

Journal: Nature Communications

Article Title: Endothelial FUNDC1 regulates metabolic reprogramming and the obesity-diabetes transition through the SIRT3/GATA2/endothelin-1 axis

doi: 10.1038/s41467-026-68548-4

Figure Lengend Snippet: a Potential KFERQ-like motifs of human FUNDC1 and SIRT3. b Interactions between HSC70 with FUNDC1 and SIRT3-L in HUVECs were detected by IP and IB. c Co-staining of FUNDC1 with HSC70 and SIRT3-L by IF. d Interaction of SIRT3-L and FUNDC1 was analyzed in HUVECs transfected with HSC70 siRNA. e Quantification of protein levels in HUVECS transfected with HSC70 siRNA. f Quantification of the binding activity of FUNDC1 with SIRT3-L in HSC70 knockdown cells, indicated by the enrichment of FUNDC1. n = 4 independent experiments. g HUVECs were transfected with control siRNA (siCtrl) or HSC70 siRNA ( siHSC70 ) in the presence of BSA or 200 µM PA. Non-nuclear fraction (Non-Nuc), nuclear fraction (Nuc), and mitochondria fraction (Mito) were prepared and subjected to immunoblotting analysis. h Quantification of protein level of SIRT3 long isoform (SIRT3-L) in the nucleus (Nuclear SIRT3-L) and in mitochondria (Mitochondrial SIRT3-L). n = 4 independent experiments. Data are presented as mean ± SD, * p < 0.05, ** p < 0.01 derived from Student’s t tests.

Article Snippet: Membranes were probed with primary antibodies against FUNDC1 (Novus, NBP1-81063), ECE1 (Santa Cruz Biotechnology, sc-376018), GATA2 (Novus, NBP1-82581; Santa Cruz Biotechnology, sc-267), AP-1 (Cell Signaling Technology, 9165), SIRT3-L (LSBio, LS- C46967 ), SIRT3-S (Cell Signaling Technology, 5490), COX IV (Cell Signaling Technology, 4850), Lamin A/C (Cell Signaling Technology, 4777), ET-1 (Santa Cruz Biotechnology, sc-517436), p-AKT Ser473 (Cell Signaling Technology, 4060), AKT (Cell Signaling Technology, 4685), β-actin (Santa Cruz Biotechnology, 47778), or GAPDH (Santa Cruz Biotechnology, 32233), and subsequently incubated with horseradish peroxidase-linked secondary antibodies.

Techniques: Staining, Transfection, Binding Assay, Activity Assay, Knockdown, Control, Western Blot, Derivative Assay

Journal: Nature Communications

Article Title: Endothelial FUNDC1 regulates metabolic reprogramming and the obesity-diabetes transition through the SIRT3/GATA2/endothelin-1 axis

doi: 10.1038/s41467-026-68548-4

Figure Lengend Snippet: Plasma and endothelium were collected from age-matched healthy donors ( n = 17) or donors with both obesity and T2DM (Obesity&T2DM, n = 19). a Characteristics of human donors. Age, body mass index (BMI), and glycohemoglobin A1c (HbA1c) levels are represented as mean ± SD. b Plasma ET-1 level in human donors. n = 17–19 donors/group. c Spearman’s correlation of plasma ET-1 level and BMI. d Spearman’s correlation of plasma ET-1 level and HbA1c. e qPCR analysis of FUNDC1 , END1 and GATA2 mRNA levels in the intima isolated from human small arterioles. n = 17–19 donors/group. f Spearman’s correlation of mRNA levels of FUNDC1 and END1 . g Spearman’s correlation of mRNA levels of GATA2 and END1 . h Spearman’s correlation of intimal FUNDC1 mRNA and plasma ET-1. i , j Human small arterioles were collected from age-matched healthy donors or donors with both obesity and T2DM (Obesity&T2DM) and subjected to western blot analysis ( i and j ). n = 6 donors/group. k , l Human femoral arteries were collected from age-matched healthy donors (n = 7) or donors with both obesity and T2DM (Obesity&T2DM; n = 5) and subjected to IHC staining. Scale bar: 50 µm. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, n.s. p > 0.05.

Article Snippet: Membranes were probed with primary antibodies against FUNDC1 (Novus, NBP1-81063), ECE1 (Santa Cruz Biotechnology, sc-376018), GATA2 (Novus, NBP1-82581; Santa Cruz Biotechnology, sc-267), AP-1 (Cell Signaling Technology, 9165), SIRT3-L (LSBio, LS- C46967 ), SIRT3-S (Cell Signaling Technology, 5490), COX IV (Cell Signaling Technology, 4850), Lamin A/C (Cell Signaling Technology, 4777), ET-1 (Santa Cruz Biotechnology, sc-517436), p-AKT Ser473 (Cell Signaling Technology, 4060), AKT (Cell Signaling Technology, 4685), β-actin (Santa Cruz Biotechnology, 47778), or GAPDH (Santa Cruz Biotechnology, 32233), and subsequently incubated with horseradish peroxidase-linked secondary antibodies.

Techniques: Clinical Proteomics, Isolation, Western Blot, Immunohistochemistry

Journal: Nature Communications

Article Title: Endothelial FUNDC1 regulates metabolic reprogramming and the obesity-diabetes transition through the SIRT3/GATA2/endothelin-1 axis

doi: 10.1038/s41467-026-68548-4

Figure Lengend Snippet: In the healthy state, endothelial FUNDC1 anchors SIRT3-L in mitochondria, limiting its nuclear accumulation and regulating its interaction with GATA2, thereby maintaining normal ET-1 production and preserving vascular and metabolic homeostasis. Under overnutrition-induced metabolic stress, SIRT3-L is released from GATA2, contributing to enhanced GATA2-mediated ET-1 transcription. Elevated ET-1 promote angiogenesis to provide the space for adipocyte hyperplasia at the early stage and later exacerbate insulin resistance and thus driving the progression from obesity to T2DM. Importantly, SIRT3-L translocation to mitochondria also facilitates FUNDC1 degradation at the early obese stage, which triggers a compensatory increase in FUNDC1 transcription and ultimately results in FUNDC1 overexpression in a late-stage. The elevation of FUNDC1 further enhances mitochondrial recruitment of SIRT3-L, amplifying ET-1 production. In contrast, in Fundc1 EC-specific knockout (ECKO) mice, the absence of FUNDC1 prevents mitochondrial recruitment of SIRT3-L, leading to its nuclear retention, where SIRT3-L promotes GATA2 degradation and suppresses ET-1 transcription. The loss of Fundc1 in ECs not only suppresses angiogenesis at early overnutrition stage but also prevents EC senescence at late stage to attenuate the potential diabetic vascular complications.

Article Snippet: Membranes were probed with primary antibodies against FUNDC1 (Novus, NBP1-81063), ECE1 (Santa Cruz Biotechnology, sc-376018), GATA2 (Novus, NBP1-82581; Santa Cruz Biotechnology, sc-267), AP-1 (Cell Signaling Technology, 9165), SIRT3-L (LSBio, LS- C46967 ), SIRT3-S (Cell Signaling Technology, 5490), COX IV (Cell Signaling Technology, 4850), Lamin A/C (Cell Signaling Technology, 4777), ET-1 (Santa Cruz Biotechnology, sc-517436), p-AKT Ser473 (Cell Signaling Technology, 4060), AKT (Cell Signaling Technology, 4685), β-actin (Santa Cruz Biotechnology, 47778), or GAPDH (Santa Cruz Biotechnology, 32233), and subsequently incubated with horseradish peroxidase-linked secondary antibodies.

Techniques: Preserving, Translocation Assay, Over Expression, Knock-Out