Journal: Investigative Ophthalmology & Visual Science

Article Title: ER Stress and Mitochondrial Perturbations Regulate Cell Death in Retinal Detachment: Exploring the Role of HIF1α

doi: 10.1167/iovs.65.11.39

Figure Lengend Snippet: Hypoxia and retinal detachment activate mitophagy markers in retinal cells. ( A ) Representative immunoblots of PINK1, Parkin and FUNDC1 proteins from 661W cells subjected to 8 hours, 18 hours, and 24 hours of hypoxia. Blots are representative of three independent experiments in each condition; α-tubulin and GAPDH were used as loading controls. ( B ) Representative immunocytochemistry images of LC3B ( green ) and FUNDC1 ( red ) stained 661W cells subjected to 24 hours of hypoxia compared to normoxic control. Scale bar : 64 µm. ( C ) Representative immunoblots of Parkin and FUNDC1 proteins from retinas of C57BL/6J mice 1 day and 3 days following retinal detachment. Blots are representative of three independent animals in each condition; α-tubulin was used as the loading control. ( D ) Relative mRNA expression of Pink1 obtained at 1 dprd and 3 dprd compared to the contralateral eye (fellow). Pum-1 was used as a housekeeping control gene for normalization. ( E ) Relative mRNA expression of Prkn obtained at 1 dprd and 3 dprd compared to the contralateral eye (fellow). Pum-1 was used as a housekeeping control gene for normalization. Bar graphs represent mean ± SEM. Statistical analysis was performed using one-way ANOVA with repeated measures followed by Tukey's test. * P < 0.05; ** P < 0.01.

Article Snippet: After fixation, the cells were permeabilized with 0.1% Triton X-100, blocked using 2% BSA, and subsequently subjected to an overnight incubation with the antibodies against FUNDC1 (1:250; Novus Biologicals) or anti-mouse LC3B (#83506, 1:250; Cell Signaling Technology).

Techniques: Western Blot, Immunocytochemistry, Staining, Control, Expressing

Journal: Investigative Ophthalmology & Visual Science

Article Title: ER Stress and Mitochondrial Perturbations Regulate Cell Death in Retinal Detachment: Exploring the Role of HIF1α

doi: 10.1167/iovs.65.11.39

Figure Lengend Snippet: HIF1α is associated with mitophagy induction after RD in retinal cells. ( A ) Representative immunoblots of HIF1α protein from 661W cells subjected to 8 hours, 18 hours, and 24 hours of hypoxia. Blots are representative of three independent experiments in each condition; α-tubulin was used as the loading control. ( B ) Representative immunoblots of HIF1α protein from retinas of C57BL/6J mice at 1 dprd and 3 dprd. Blots are representative of three independent animals in each condition; α-tubulin was used as the loading control. ( C ) Relative mRNA expression of Hspa5 obtained from retinas of Rho/Cre + and HIF1αΔrod mice at 3 dprd compared to the contralateral eye (fellow). Pum-1 was used as a housekeeping control gene for normalization. ( D ) Relative mRNA expression of Ddit3 obtained from retinas of Rho/Cre + and HIF1αΔrod mice at 3 dprd compared to the contralateral eye (fellow). Pum-1 was used as a housekeeping control gene for normalization. ( E ) Relative mRNA expression of Pgc1a and Tfam obtained from retinas of Rho/Cre + and HIF1αΔrod mice at 3 dprd compared to the contralateral eye (fellow). Pum-1 was used as a housekeeping control gene for normalization. ( F ) Representative immunoblots of Parkin and FUNDC1 proteins from retinas of Rho/Cre + and HIF1αΔrod mice at 3 dprd compared to the contralateral eye (fellow). GAPDH and α-tubulin were used as the loading control. ( G ) Relative mRNA expression of Prkn obtained from retinas of Rho/Cre+ and HIF1αΔrod mice at 3 dprd compared to the contralateral eye (fellow). Pum-1 was used as a housekeeping control gene for normalization. ( H ) Relative mRNA expression of Pink obtained from retinas of Rho/Cre + and HIF1αΔrod mice at 3 dprd compared to the contralateral eye (fellow). Pum-1 was used as a housekeeping control gene for normalization. Bar graphs represent mean ± SEM. Statistical analysis was performed using two-way ANOVA with repeated measures followed by Tukey's test. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: After fixation, the cells were permeabilized with 0.1% Triton X-100, blocked using 2% BSA, and subsequently subjected to an overnight incubation with the antibodies against FUNDC1 (1:250; Novus Biologicals) or anti-mouse LC3B (#83506, 1:250; Cell Signaling Technology).

Techniques: Western Blot, Control, Expressing

Journal: The American journal of pathology

Article Title: Molecular and functional alterations in a mouse cardiac model of Friedreich ataxia: activation of the integrated stress response, eIF2α phosphorylation, and the induction of downstream targets.

doi: 10.1016/j.ajpath.2013.05.032

Figure Lengend Snippet: Figure 6 Proposed pathogenesis of heart failure in FA. Frataxin defi- ciency results in the early activation of the ISR via phosphorylation (P) of eIF2a and induction of its downstream stress-inducible molecules, including Atf4 and Chop. The precise stress stimulus for the ISR activation is unclear, but may involve early and pronounced perturbation in heme synthesis (markedly reduced Fech expression) acting through Hri in KO mice, compared to WT mice. In addition, the later involvement of ER stress (up-regulation of Bip and cleaved caspase-12) could potentiate the prolonged activation of the ISR pathway. ISR activation subsequently enhances autophagy (up- regulation of Atg3, LC3-II, p62, and Fundc1) and apoptosis (increased expression of Chop, Bax and Bcl-2, and cleaved caspase-12), leading to cardiomyocyte death. Histological alterations are first apparent from 5 weeks of age with iron accumulation, followed by fibrosis and cardiomyocyte degeneration from 6 weeks of age. Severe progressive reduction in cardiac function ensues, resulting in heart failure. Casp12, caspase-12.

Article Snippet: Protein isolation and Western blot analysis were performed using established techniques.3 The primary antibodies used were against TfR1 (13-6890; Life Technologies), Atf4 (sc-200; SantaCruzBiotechnology,SantaCruz,CA),Mthfd2 (ab37840; Abcam, Cambridge, UK), Asns (1732-1; Epitomics, Burlingame,CA), Chop (SC-575; Santa Cruz), p-eIF2a (Ser51; 3398; Cell Signaling Technology, Danvers, MA), eIF2a (2103; Cell Signaling Technology), Atg3 (3415; Cell Signaling Technology), LC3 (PD014; MBL International, Woburn, MA), p62 (GP62-C; Progen Biotechnik, Heidelberg, Germany), caspase12 (2202; Cell Signaling Technology), Bax (2772; Cell Signaling Technology), Bcl-2 (2870; Cell Signaling Technology), Itgb1bp3 (K0099-3; MBL International), p-Perk (Thr980; 3179; Cell Signaling Technology), Perk (3192; Cell Signaling Technology), Bip (3177; Cell Signaling Technology), Hri (07-728; Millipore, Sydney, Australia), Fech (from Harry Dailey, University of Georgia30), Fundc1 (ARP53280_P050; Aviva Systems Biology, San Diego, CA), and Gapdh (SC-25778; Santa Cruz Biotechnology).

Techniques: Activation Assay, Phospho-proteomics, Expressing

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: The aortic sections were incubated with FUNDC1 (Biorbyt, orb156964), 8-OHdG (abcam, ab62623), or ET-1 (Novus, NB300-526) antibody at 4 °C overnight and followed by incubation with EnVision® + Dual Link System-HRP (Dako Cytomation, 3468) and the signal was visualized using the Liquid DAB+ Substrate Chromogen System and imaged using an inverted phase contrast microscope (Nikon, Tokyo, Japan).

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: The aortic sections were incubated with FUNDC1 (Biorbyt, orb156964), 8-OHdG (abcam, ab62623), or ET-1 (Novus, NB300-526) antibody at 4 °C overnight and followed by incubation with EnVision® + Dual Link System-HRP (Dako Cytomation, 3468) and the signal was visualized using the Liquid DAB+ Substrate Chromogen System and imaged using an inverted phase contrast microscope (Nikon, Tokyo, Japan).

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: The aortic sections were incubated with FUNDC1 (Biorbyt, orb156964), 8-OHdG (abcam, ab62623), or ET-1 (Novus, NB300-526) antibody at 4 °C overnight and followed by incubation with EnVision® + Dual Link System-HRP (Dako Cytomation, 3468) and the signal was visualized using the Liquid DAB+ Substrate Chromogen System and imaged using an inverted phase contrast microscope (Nikon, Tokyo, Japan).

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: The aortic sections were incubated with FUNDC1 (Biorbyt, orb156964), 8-OHdG (abcam, ab62623), or ET-1 (Novus, NB300-526) antibody at 4 °C overnight and followed by incubation with EnVision® + Dual Link System-HRP (Dako Cytomation, 3468) and the signal was visualized using the Liquid DAB+ Substrate Chromogen System and imaged using an inverted phase contrast microscope (Nikon, Tokyo, Japan).

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: The aortic sections were incubated with FUNDC1 (Biorbyt, orb156964), 8-OHdG (abcam, ab62623), or ET-1 (Novus, NB300-526) antibody at 4 °C overnight and followed by incubation with EnVision® + Dual Link System-HRP (Dako Cytomation, 3468) and the signal was visualized using the Liquid DAB+ Substrate Chromogen System and imaged using an inverted phase contrast microscope (Nikon, Tokyo, Japan).

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: The aortic sections were incubated with FUNDC1 (Biorbyt, orb156964), 8-OHdG (abcam, ab62623), or ET-1 (Novus, NB300-526) antibody at 4 °C overnight and followed by incubation with EnVision® + Dual Link System-HRP (Dako Cytomation, 3468) and the signal was visualized using the Liquid DAB+ Substrate Chromogen System and imaged using an inverted phase contrast microscope (Nikon, Tokyo, Japan).

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: The aortic sections were incubated with FUNDC1 (Biorbyt, orb156964), 8-OHdG (abcam, ab62623), or ET-1 (Novus, NB300-526) antibody at 4 °C overnight and followed by incubation with EnVision® + Dual Link System-HRP (Dako Cytomation, 3468) and the signal was visualized using the Liquid DAB+ Substrate Chromogen System and imaged using an inverted phase contrast microscope (Nikon, Tokyo, Japan).

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: The aortic sections were incubated with FUNDC1 (Biorbyt, orb156964), 8-OHdG (abcam, ab62623), or ET-1 (Novus, NB300-526) antibody at 4 °C overnight and followed by incubation with EnVision® + Dual Link System-HRP (Dako Cytomation, 3468) and the signal was visualized using the Liquid DAB+ Substrate Chromogen System and imaged using an inverted phase contrast microscope (Nikon, Tokyo, Japan).

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: The aortic sections were incubated with FUNDC1 (Biorbyt, orb156964), 8-OHdG (abcam, ab62623), or ET-1 (Novus, NB300-526) antibody at 4 °C overnight and followed by incubation with EnVision® + Dual Link System-HRP (Dako Cytomation, 3468) and the signal was visualized using the Liquid DAB+ Substrate Chromogen System and imaged using an inverted phase contrast microscope (Nikon, Tokyo, Japan).

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

Journal: Science signaling

Article Title: The mitophagy effector FUNDC1 controls mitochondrial reprogramming and cellular plasticity in cancer cells

doi: 10.1126/scisignal.aaz8240

Figure Lengend Snippet: (A) PC3 cells transfected with control non-targeting siRNA (siCtrl) or FUNDC1-directed pooled siRNA (siFND1) were labeled with Talin-RFP and analyzed for focal adhesion (FA) dynamics by time-lapse videomicroscopy. Representative images at 0 h and 2 h from 3 independent experiments are shown. (B) The rate (events/h) of FA assembly (top) or disassembly (bottom) was quantified from the cells in (A). Each point corresponds to FA events in an individual cell (11–14 cells per condition). Mean±SD (N=3 independent experiments per group). *, p=0.02–0.04. (C and D) PC3 cells transfected with siCtrl or siFND1 were analyzed for cell motility in 2D contour plots by time-lapse video microscopy (C) with quantification of speed of cell movements (D, top) and distance traveled by individual cells (D, bottom). Each tracing in (C) corresponds to the movements of an individual cell (49 cells per condition) in a representative experiment. The cutoff velocities for slow (blue, <0.6 μm/min)- or fast (orange, >0.6 μm/min)-moving cells are indicated. Mean±SD (N=3 independent experiments per group). ***, p<0.0001. (E) PC3 cells transfected with siCtrl or siFND1 were analyzed for 2D chemotaxis in a rose plot (top) with quantification of the forward migration index (bottom). Arrows indicate the direction of the chemotactic gradient. Each point corresponds to an individual cell (89 cells per condition; N=3 independent experiments per group). (F) PC3 cells transfected with siCtrl or siFND1 were analyzed for invasion across Matrigel-coated Transwell inserts. Each point corresponds to an individual determination. Mean±SD (N=3 independent experiments per group). ***, p=0.0001. (G) The indicated prostate cancer cell lines were transfected with vector or FND1 cDNA and analyzed for Matrigel invasion. Mean±SD (N=3 independent experiments per group). **, p=0.001; ***, p <0.0001 – 0.0005. (H and I) PC3 cells transfected with vector or FND1 cDNA were analyzed by Western blotting (H, representative blot of three independent experiments) and the intensity of phosphorylated (p) FAK protein band was quantified by densitometry (I). Mean±SD (N=3 independent experiments per group). ***, p=0.0008. (J) PC3 cells transfected with siCtrl or siFND1 were analyzed for Matrigel invasion in the presence or absence of FAK-directed siRNA (siFAK). Mean±SD (N=3 independent experiments per group). ***, p<0.0001.

Article Snippet: Antibodies to FUNDC1 (Aviva System Biology, diluted 1:500), Drp1 (clone D6C7, Cell Signaling cat. #8570 diluted 1:1000), Ser 616 -phosphorylated Drp1 (Cell Signaling cat. #3455 diluted 1:1000), Drp1 (Cell Signaling cat. #5391 diluted 1:1000), Tyr 925 -phosphorylated FAK (Cell Signaling cat. #3284 diluted 1:1000), FAK (Cell Signaling cat. n. #3285), FLAG (clone M-2, Sigma cat. #F1804 diluted 1:5000) and β-actin (clone AC-15, Sigma cat. #A5441 diluted 1:100000) were used for Western blotting.

Techniques: Transfection, Labeling, Microscopy, Chemotaxis Assay, Migration, Plasmid Preparation, Western Blot

Journal: Science signaling

Article Title: The mitophagy effector FUNDC1 controls mitochondrial reprogramming and cellular plasticity in cancer cells

doi: 10.1126/scisignal.aaz8240

Figure Lengend Snippet: (A) PC3 cells were fractionated in submitochondrial compartments and analyzed by Western blotting. MTE, mitochondrial extracts; OM, outer membrane; IM, inner membrane; IMS, intermembrane space; Mat, matrix. Data are representative of 2 independent experiments. (B) Ingenuity pathway analysis of a mitochondrial FUNDC1 interactome identified by LC-MS/MS proteomics (N=1 independent experiment). The fold-induction for FUNDC1-associated proteins compared to Flag-vector are indicated with red color intensity. (C) PC3 cells transfected with Flag-vector or Flag-FND1 cDNA were immunoprecipitated (IP) with an antibody to Flag and immune complexes were analyzed by Western blotting. Data are representative of 2 independent experiments. WCE, whole cell extracts. (D) PC3 cells were immunoprecipitated with non-binding IgG or an antibody to endogenous ATP5C1 and immune complexes were analyzed by Western blotting. Data are representative of 2 independent experiments. WCE, whole cell extracts. (E) PC3 cells transfected with siCtrl or siFND1 were reconstituted with FND1 cDNA and analyzed for complex V (C.V) activity. Tracings from two independent experiments (Exp) are shown. Mean±SD of 3 technical replicates per experiment. (F) Citrate synthase-normalized complex V (C.V) activity as in (E) was quantified. Two independent experiments (Exp) are shown. Mean±SD of 3 technical replicates per experiment. (G) PC3 cells stably transduced with pLKO or shFND1 were reconstituted with Flag-LonP1 cDNA, and Flag-eluted LonP1 immune complexes (Elu) (top, representative blot of 3 independent experiments) were analyzed for LonP1 proteolytic activity (bottom). TCE, total cell extracts. Mean±SD (N=3 independent experiments per group). (H and I) PC3 cells transfected with siCtrl or siFND1 were reconstituted with LonP1 cDNA, extracted at increasing concentrations of NP-40 and detergent-insoluble protein bands corresponding to complex V subunits, ATP5C1, ATP5O and ATP5B were analyzed by Western blotting (H, representative blot of 2 independent experiments) and quantified by densitometry (I). VDAC was a control. Two independent experiments (Exp) are shown (I).

Article Snippet: Antibodies to FUNDC1 (Aviva System Biology, diluted 1:500), Drp1 (clone D6C7, Cell Signaling cat. #8570 diluted 1:1000), Ser 616 -phosphorylated Drp1 (Cell Signaling cat. #3455 diluted 1:1000), Drp1 (Cell Signaling cat. #5391 diluted 1:1000), Tyr 925 -phosphorylated FAK (Cell Signaling cat. #3284 diluted 1:1000), FAK (Cell Signaling cat. n. #3285), FLAG (clone M-2, Sigma cat. #F1804 diluted 1:5000) and β-actin (clone AC-15, Sigma cat. #A5441 diluted 1:100000) were used for Western blotting.

Techniques: Western Blot, Liquid Chromatography with Mass Spectroscopy, Plasmid Preparation, Transfection, Immunoprecipitation, Binding Assay, Activity Assay, Stable Transfection, Transduction

Journal: Science signaling

Article Title: The mitophagy effector FUNDC1 controls mitochondrial reprogramming and cellular plasticity in cancer cells

doi: 10.1126/scisignal.aaz8240

Figure Lengend Snippet: (A and B) PC3 (top), C42B (middle) or DU145 (bottom) cells were transfected with vector or FND1 cDNA and analyzed for Matrigel invasion (A) or cell proliferation by direct cell counting (B). Mean±SD (N=3 or 5 independent experiments per condition). **, p=0.001; ***, p <0.0001–0.0005. (C and D) PC3 cells stably transduced with pLKO or FUNDC1-directed shRNA (shFND1) were analyzed for colony formation (C) and quantified after 14 d (D). Mean±SD (N=3 independent experiments per group). ***, p<0.0001. (E) PC3 cells transduced with pLKO or shFND1 were engrafted subcutaneously onto the flanks of immunocompromised mice (N=10 animals/group) and tumor growth was quantified at the indicated time intervals. Each line corresponds to an individual tumor. The mean±SD tumor size (mm3) for each animal group harvested at day 27 is indicated, p<0.0001. (F) Representative tumor samples from each animal group in (E) were excised at the end of the experiment and analyzed for Ki67 staining by immunohistochemistry. Representative images are shown. Scale bar, 100 μm. (G and H) Representative lung samples isolated from each animal group in (E) were stained with an antibody to human mitochondria by immunohistochemistry (G, representative images) and the number of lung metastatic foci was quantified (H). Scale bar, 100 μm. Mean±SD (N=25 determinations per group). ***, p<0.0001. (I) PC3 cells stably transduced with pLKO or shFND1 were injected into the spleen of immunocompromised mice (N=5 animals per group) and metastatic foci to the liver were quantified after 11 days by histology. Mean±SD (N=8–10 determinations per group). ***, p=0.0009. (J and K) RNA-seq expression data from 33 cancer types (TCGA) for genes (Spearman r>0.2; p<0.05) positively (J, GO:0046034, ATP metabolism) or negatively (K, GO:0000302, response to ROS; GO:0048870, cell motility; GO:0008283, cell proliferation) correlated with FUNDC1 (FND1) expression.

Article Snippet: Antibodies to FUNDC1 (Aviva System Biology, diluted 1:500), Drp1 (clone D6C7, Cell Signaling cat. #8570 diluted 1:1000), Ser 616 -phosphorylated Drp1 (Cell Signaling cat. #3455 diluted 1:1000), Drp1 (Cell Signaling cat. #5391 diluted 1:1000), Tyr 925 -phosphorylated FAK (Cell Signaling cat. #3284 diluted 1:1000), FAK (Cell Signaling cat. n. #3285), FLAG (clone M-2, Sigma cat. #F1804 diluted 1:5000) and β-actin (clone AC-15, Sigma cat. #A5441 diluted 1:100000) were used for Western blotting.

Techniques: Transfection, Plasmid Preparation, Cell Counting, Stable Transfection, Transduction, shRNA, Staining, Immunohistochemistry, Isolation, Injection, RNA Sequencing Assay, Expressing

Journal: Science signaling

Article Title: The mitophagy effector FUNDC1 controls mitochondrial reprogramming and cellular plasticity in cancer cells

doi: 10.1126/scisignal.aaz8240

Figure Lengend Snippet: (A) Heatmap of changes in metabolite levels in PC3 cells transfected with siCtrl or siFND1. Fold changes, p values and false discovery rate (FDR) are indicated. Data are from a representative experiment (siCtrl) or two independent experiments in triplicate (siFND1, 1A-C; 2A-C). (B) Schematic diagram of mitochondrial bioenergetics and ROS pathways affected by FUNDC1 silencing as in (A). (C) PC3 cells transfected with siCtrl or siFND1were analyzed for oxygen consumption rates (OCR) on a Seahorse XFe96 Bioenergetics Flux Analyzer. Tracings from two independent experiments (Exp) are shown. Mean±SD of 3 technical replicates per experiment. (D) PC3 cells transfected with siCtrl or siFND1were analyzed for MitoSox reactivity and mitochondrial membrane potential (TMRE) by flow cytometry. Mean±SD (N=3–4 independent experiments per group). **, p=0.001. (E) PC3 cells transfected with siCtrl or siFND1were analyzed for MitoSox reactivity in the presence or absence of the superoxide scavenger, MnTBAP, by flow cytometry. Mean±SD (N=3 independent experiments per group). **, p=0.005; *, p=0.03. (F) PC3 cells transfected with siCtrl or siFND1were analyzed for speed of mitochondrial movements (top) or distance traveled by individual mitochondria (bottom) with or without MnTBAP. Each symbol corresponds to the tracked movement of an individual mitochondrion. Data are representative of 3 independent experiments. The mean±SD of mitochondrial speed (siCtrl compared to siFND1, p<0.0001; siFND1 compared to siFND1+MnTBAP, p=0.002) and distance traveled (siCtrl compared to siFND1, p=0.001; siFND1 compared to siFND1+MnTBAP, p=0.0005) are indicated. (G) siCtrl- or siFND1-transfected PC3 cells were analyzed for cellular motility in 2D contour plots in the presence or absence of MnTBAP. Each tracing corresponds to the movements of an individual cell. The cut-off velocities for slow (<0.25 μm/min)- or fast (>0.25 μm/min)-moving cells are indicated. Data are representative of 3 independent experiments per group. The speed (μm/min) of cell motility (siCtrl compared to siFND1, p<0.0001; siFND1 compared to siFND1+MnTBAP, p=0.006) and distance (μm) traveled (siCtrl compared to siFND1, p<0.0001; siFND1 compared to siFND1+MnTBAP, p=0.006) are indicated. (H) PC3 cells transfected with siCtrl or siFND1 were analyzed for Matrigel invasion in the presence or absence of MnTBAP. Mean±SD (N=3 independent experiments per group). ***, p<0.0001. (I) PC3 cells transfected with siCtrl or siFND1 were analyzed for cell proliferation in the presence or absence of MnTBAP by direct cell counting. Mean±SD (N=4 independent experiments per group). **, p=0.03.

Article Snippet: Antibodies to FUNDC1 (Aviva System Biology, diluted 1:500), Drp1 (clone D6C7, Cell Signaling cat. #8570 diluted 1:1000), Ser 616 -phosphorylated Drp1 (Cell Signaling cat. #3455 diluted 1:1000), Drp1 (Cell Signaling cat. #5391 diluted 1:1000), Tyr 925 -phosphorylated FAK (Cell Signaling cat. #3284 diluted 1:1000), FAK (Cell Signaling cat. n. #3285), FLAG (clone M-2, Sigma cat. #F1804 diluted 1:5000) and β-actin (clone AC-15, Sigma cat. #A5441 diluted 1:100000) were used for Western blotting.

Techniques: Transfection, Flow Cytometry, Cell Counting