antibody against fundc1  (Cell Signaling Technology Inc)

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: International Journal of Biological Sciences

Article Title: Emodin Inhibits NLRP3 Inflammasome Activation and Protects Against Sepsis via Promoting FUNDC1-Mediated Mitophagy

doi: 10.7150/ijbs.110904

Figure Lengend Snippet: Emodin suppresses NLRP3 inflammasome activation via FUNDC1-mediated mitophagy. (A) Cell lysates of LPS-primed THP-1 with or without nigericin incubated with emodin-Sepharose. The pull-down samples and input were analyzed by western blot. (B) Effect of emodin on the ATPase activity of CK2. After incubation CK2 plus indicated different concentrations of free emodin (12.5 μM, 25 μM, and 50μM), ATP was measured by CellTiter-Glo Assay Kit and normalized to the control. (C) THP-1 cells, primed with LPS, were exposed to emodin or a vehicle control, followed by nigericin stimulation. Western blot analysis was conducted to assess levels of LC3II, FUNDC1, and p-FUNDC1. (D) THP-1 cells were transfected with Flag-tagged FUNDC1 and subjected to the same treatment as in (C). Confocal microscopy was used to examine the colocalization of LC3 (red) and Flag-FUNDC1 (green). Scale bars: 10 μm. (E) Cell Lys underwent immunoprecipitation with an anti-LC3 antibody, and subsequent western blot was performed to evaluate the interaction between LC3 and FUNDC1 in THP-1 cells treated as in (C). (D-F) THP-1 cells were transfected with FUNDC1 siRNA or a negative control siRNA for 24 h, followed by the treatment regimen outlined in the Figure. Western blot analysis was employed to measure FUNDC1 expression in Lys (D). Confocal microscopy was used to visualize the colocalization of LC3 (red) and Mitotracker (green). Scale bars: 10 μm (E). Western blot analysis was conducted to detect IL-1β (p17), caspase-1 (p20) in the Sup and pro-IL-1β (p30), caspase-1 (p45), LC3II in Lys (F). The data presented are representative of three independent experiments. ** P <0.01 compared to control. *** P <0.001 compared to control. ns , not significant.

Article Snippet: ABclonal (Wuhan, China) supplied antibodies against FUNDC1 (1:1000, a22001), LC3B (1:1000, a196665), and Flag (1:1000, AE005), as well as secondary antibodies: ABflo 488-conjugated goat anti-mouse IgG (1:1000, AS037), ABflo 555-conjugated goat anti-mouse IgG (1:1000, AS057), and ABflo Cy3-conjugated goat anti-rabbit IgG (1:1000, AS007).

Techniques: Activation Assay, Incubation, Western Blot, Activity Assay, Glo Assay, Control, Transfection, Confocal Microscopy, Immunoprecipitation, Negative Control, Expressing

Journal: International Journal of Biological Sciences

Article Title: Emodin Inhibits NLRP3 Inflammasome Activation and Protects Against Sepsis via Promoting FUNDC1-Mediated Mitophagy

doi: 10.7150/ijbs.110904

Figure Lengend Snippet: Emodin's effects on LPS-induced sepsis rely on FUNDC1-mediated mitophagy. (A) Schematic representation of the AAV-F4/80p-EGFP-MIR155(RNAi)-SV40-PolyA construct (AAV-shFUNDC1). (B) Experimental design overview: Mice were administered tail vein injections of either AAV-shFUNDC1 or AAV-Ctrl-shRNA. After 21 days, mice were pretreated with emodin (20 mg/kg), followed by an intraperitoneal injection of LPS (10 mg/kg) one hour later. (C) Flow cytometry was used to evaluate GFP expression in lung macrophages from mice infected with AAV-Ctrl-shRNA or AAV-shFUNDC1 on day 21. (D) Western blot analysis of FUNDC1 expression in lung tissue from mice infected with AAV-shFUNDC1 or AAV-Ctrl-shRNA. (E) Survival rates of male C57BL/6 mice were tracked over 72 hours. Statistical significance was assessed using the log-rank test. (F-G) IL-1β and TNF-α levels in serum (F) and PLF (G) were quantified by ELISA. (H-I) Representative H&E-stained images of lung (H) and liver (I) tissues. Scale bar: 100 μm. (J) Western blot analysis was conducted to measure the expression levels of NLRP3, pro-caspase-1 (p45), caspase-1 (p20), FUNDC1, and LC3II proteins in lung tissue. Data are expressed as mean ± SEM. Results are representative of at least two independent experiments, with 5-7 animals per group. * P <0.05 compared to control. ** P <0.01 compared to control. *** P <0.001 compared to control. ns , not significant.

Article Snippet: ABclonal (Wuhan, China) supplied antibodies against FUNDC1 (1:1000, a22001), LC3B (1:1000, a196665), and Flag (1:1000, AE005), as well as secondary antibodies: ABflo 488-conjugated goat anti-mouse IgG (1:1000, AS037), ABflo 555-conjugated goat anti-mouse IgG (1:1000, AS057), and ABflo Cy3-conjugated goat anti-rabbit IgG (1:1000, AS007).

Techniques: Construct, shRNA, Injection, Flow Cytometry, Expressing, Infection, Western Blot, Enzyme-linked Immunosorbent Assay, Staining, Control

Journal: Stroke and Vascular Neurology

Article Title: Src inhibition rescues FUNDC1-mediated neuronal mitophagy in ischaemic stroke

doi: 10.1136/svn-2023-002606

Figure Lengend Snippet: Knockout of FUNDC1 has no effect on short-term outcomes of ischaemic stroke. (A) Cerebral blood flow (CBF) of WT and FUNDC1 –/– mice. (B) Quantification for the variation of CBF at each time point of the perioperative period. n=5 mice for each genotype. (C) Representative TTC staining of brains from WT and FUNDC1 –/– mice subjected to tMCAO at 24 hours after surgery. (D) Quantification for the infarcted volumes. n=10 mice for each genotype. (E) Degenerated neurons in the infarcted brains at 24 hours after reperfusion. (F) Quantification for the percentage of degenerated neurons. n=6 mice for each genotype. (G) Apoptosis in ipsilateral and contralateral brain tissues from WT and FUNDC1 –/– mice subjected to tMCAO were detected by western blotting at 24 hours after reperfusion. (H) Semi-quantification for apoptosis-associated protein PARP, cleaved PARP, caspase 9, cleaved caspase 9, caspase 3 and cleaved caspase 3. n=6 mice for each group. (I) Comparison for neurological deficits including Bederson score (left) and Grip test (right). n=22 mice for each genotype. (J) Χ 2 test fourfold tables for 24-hour death events in WT and FUNDC1 –/– mice. DAPI: 4',6-diamidino-2-phenylindole; FJC, Fluore Jade C; FUNDC1, FUN14 domain-containing 1; KO: Knockout; PARP: poly ADP-ribose polymerase; tMCAO, transient middle cerebral artery occlusion; TTC, triphenyltetrazolium chloride; WT, wild-type.

Article Snippet: At the end of reperfusion, cells were fixed with 4% PFA and incubated with an antibody against FUNDC1 (1:100; CST, 49240).

Techniques: Knock-Out, Staining, Western Blot, Comparison

Journal: Stroke and Vascular Neurology

Article Title: Src inhibition rescues FUNDC1-mediated neuronal mitophagy in ischaemic stroke

doi: 10.1136/svn-2023-002606

Figure Lengend Snippet: Absence of FUNDC1 does not influence long-term outcomes of ischaemic stroke. (A) 14-day survival rate after tMCAO. n=10 mice for each genotype. (B–C) Body weight recovery (B) and behavioural tests (C) including Garcia score, mNSS score, rotarod test, hanging test were assessed before the surgery (WT, n=10; FUNDC1 −/− , n=10) and at 1 day (WT, n=10; FUNDC1 −/− , n=8), 3-day (WT, n=7; FUNDC1 −/− , n=7), 7-day (WT, n=6; FUNDC1 −/− , n=6), 14-day (WT, n=5; FUNDC1 −/− , n=5) after tMCAO. FUNDC1, FUN14 domain-containing 1; mNSS, Modified Neurological Severity Score; tMCAO, transient middle cerebral artery occlusion; WT, wild-type.

Article Snippet: At the end of reperfusion, cells were fixed with 4% PFA and incubated with an antibody against FUNDC1 (1:100; CST, 49240).

Techniques: Modification

Journal: Stroke and Vascular Neurology

Article Title: Src inhibition rescues FUNDC1-mediated neuronal mitophagy in ischaemic stroke

doi: 10.1136/svn-2023-002606

Figure Lengend Snippet: Deletion of FUNDC1 does not affect neuronal mitophagy in vivo. (A) Time course of mitophagy in vivo was detected by western blotting. (B) Semi-quantification for western blotting detection in panel A. n=3 mice for each time point. (C) WT and FUNDC1 −/− mice were subjected to sham surgery or tMCAO for 24 hours. Mitophagy was detected by western blotting. (D) Semi-quantification for western blotting detection in panel C. n=6 mice per group. (E) Brain sections from both genotypes of normal mice or mice with stroke were labelled with NeuN (red), LC3 (green), and Tomm20 (magenta). (F) Quantification of the overlap coefficient of LC3 and Tomm20. n=6 mice per group. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. FUNDC1, FUN14 domain-containing 1; KO: Knockout; LC3, light chain 3; SQSTM1: Sequestosome 1; tMCAO, transient middle cerebral artery occlusion; WT, wild-type.

Article Snippet: At the end of reperfusion, cells were fixed with 4% PFA and incubated with an antibody against FUNDC1 (1:100; CST, 49240).

Techniques: In Vivo, Western Blot, Knock-Out

Journal: Stroke and Vascular Neurology

Article Title: Src inhibition rescues FUNDC1-mediated neuronal mitophagy in ischaemic stroke

doi: 10.1136/svn-2023-002606

Figure Lengend Snippet: Loss of FUNDC1 does not affect neuronal mitophagy and mitochondrial qualities in vitro. (A) Time course of neuronal mitophagy in vitro was detected by western blotting. (B) Semi-quantification for western blotting detection in panel A. n=3 for independent experiments. (C) Cortical neurons isolated from WT and FUNDC1 –/– mice were subjected to control or OGD/R treatment for 9 hours. Mitophagy was detected by western blotting. (D) Semi-quantification for western blotting detection in panel C. n=3 per independent experiment. (E) Normal-cultured or OGD/R-treated neurons with two genotypes were labelled with MitoTracker (red) and GFP-LC3 (green), then visualised by confocal microscopy. (F) Quantification of the number of LC3 puncta per cell (left) and colocalisation coefficient of mitochondria and LC3 puncta (right). n=3 for independent experiment. **p<0.01, ***p<0.001, ****p<0.0001. FUNDC1, FUN14 domain-containing 1; GFP: Green Fluorescent Protein; KO: Knockout; LC3, light chain 3; OGD/R, oxygen glucose deprivation/reperfusion; SQSTM1: Sequestosome 1; WT, wild-type.

Article Snippet: At the end of reperfusion, cells were fixed with 4% PFA and incubated with an antibody against FUNDC1 (1:100; CST, 49240).

Techniques: In Vitro, Western Blot, Isolation, Control, Cell Culture, Confocal Microscopy, Knock-Out

Journal: Stroke and Vascular Neurology

Article Title: Src inhibition rescues FUNDC1-mediated neuronal mitophagy in ischaemic stroke

doi: 10.1136/svn-2023-002606

Figure Lengend Snippet: FUNDC1 is inactivated in later stages of neuronal I/R injury. (A) Time course of FUNDC1 phosphorylation was detected by western blotting in vivo. (B) Semi-quantification for p-Tyr18 and total FUNDC1 in panel A. n=3 mice per time point. (C) Immunostaining of p-FUNDC1 (green) in neurons (red). (D) Quantification for proportion of p-FUNDC1 positive neurons. n=6 mice per time point. (E) Time course of FUNDC1 phosphorylation was detected by western blotting in vitro. n=3 for independent experiments. (F) Semi-quantification for LC3 II interacted with FUNDC1 as is shown in panel G. n=3 for independent experiments. (G) Isolated cortical neurons were subjected to 2 hours of OGD, followed by 0 hours, 6 hours, 9 hours or 12 hours of reperfusion. Interactions between FUNDC1 and LC3 were detected by co-immunoprecipitation. (H) Fluorescent staining of FUNDC1 (red) and GFP-LC3 (green) in isolated neurons subjected to control treatment, OGD/R 0 hours, or OGD/R 9 hours. (I) Quantification of the number of LC3 puncta per cell (left) and percentage of LC3 puncta colocalised with FUNDC1 (right). N=3 for independent experiment. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. FUNDC1, FUN14 domain-containing 1; Green Fluorescent Protein; I/R, ischaemia/reperfusion; LC3, light chain 3; OGD/R, oxygen glucose deprivation/reperfusion; tMCAO, transient middle cerebral artery occlusion; WT, wild-type.

Article Snippet: At the end of reperfusion, cells were fixed with 4% PFA and incubated with an antibody against FUNDC1 (1:100; CST, 49240).

Techniques: Phospho-proteomics, Western Blot, In Vivo, Immunostaining, In Vitro, Isolation, Immunoprecipitation, Staining, Control

Journal: Stroke and Vascular Neurology

Article Title: Src inhibition rescues FUNDC1-mediated neuronal mitophagy in ischaemic stroke

doi: 10.1136/svn-2023-002606

Figure Lengend Snippet: Src is activated in later stages of neuronal ischaemia/reperfusion injury. (A) Src phosphorylation in vivo at different time points was detected by western blotting. (B) Semi-quantification for p-Tyr416 and total Src in panel A. n=3 mice per time point. (C) Immunostaining of p-Src (green) in neurons (red). (D) Quantification for proportion of p-Src positive neurons. n=6 mice per time point. (E) Src phosphorylation in vitro at different time points was detected by western blotting. n=3 for independent experiments. (F) Semi-quantification for Src interacted with FUNDC1 as is shown in panel G. n=3 for independent experiments. (G) Time course of FUNDC1-Src interaction in isolated neurons were detected by co-immunoprecipitation. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. FUNDC1, FUN14 domain-containing 1; OGD, oxygen glucose deprivation; tMCAO, transient middle cerebral artery occlusion.

Article Snippet: At the end of reperfusion, cells were fixed with 4% PFA and incubated with an antibody against FUNDC1 (1:100; CST, 49240).

Techniques: Phospho-proteomics, In Vivo, Western Blot, Immunostaining, In Vitro, Isolation, Immunoprecipitation

Journal: Stroke and Vascular Neurology

Article Title: Src inhibition rescues FUNDC1-mediated neuronal mitophagy in ischaemic stroke

doi: 10.1136/svn-2023-002606

Figure Lengend Snippet: Pharmacological inhibition of Src rescues FUNDC1-mediated mitophagy in neurons subjected to ischaemia/reperfusion injury. (A) Neurons subjected to OGD/R were treated with vehicle or PP1, and were labelled using antibodies against FUNDC1 (red) and GFP-LC3 (green). (B) Quantification of the number of LC3 puncta per cell (left) and percentage of LC3 puncta colocalised with FUNDC1 (right). n=3 for independent experiment. (C) Mitophagy in mice with both genotypes treated by PP1 in brain samples was detected by western blotting. (D) Semi-quantification for western blotting detection in panel C. n=6 mice per group. (E) Representative TTC staining of brains from vehicle-treated WT mice (n=5), FUNDC1 –/– mice (n=5), PP1-treated WT mice (n=6), and PP1-treated FUNDC1 –/– mice (n=6). (F) Quantification of infarcted volume. (G) Comparison of neurological deficits among vehicle-treated WT mice (n=11), FUNDC1 –/– mice (n=11) PP1-treated WT mice (n=12), and PP1-treated FUNDC1 –/– mice (n=12). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. FUNDC1, FUN14 domain-containing 1; GFP: Green Fluorescent Protein; OGD/R, oxygen glucose deprivation/reperfusion; KO: Knockout; LC3, light chain 3; SQSTM1: Sequestosome 1; tMCAO, transient middle cerebral artery occlusion; WT, wild-type.

Article Snippet: At the end of reperfusion, cells were fixed with 4% PFA and incubated with an antibody against FUNDC1 (1:100; CST, 49240).

Techniques: Inhibition, Western Blot, Staining, Comparison, Knock-Out

Journal: Stroke and Vascular Neurology

Article Title: Src inhibition rescues FUNDC1-mediated neuronal mitophagy in ischaemic stroke

doi: 10.1136/svn-2023-002606

Figure Lengend Snippet: Working model of FUNDC1 inactivation by Src during neuronal I/R injury (created with BioRender.com). Although neuronal I/R induces mitophagy, hyperactivated Src inactivates FUNDC1, leading to FUNDC1 exclusion from mitophagy. Even in the absence of FUNDC1, neurons can initiate mitophagy through the BNIP3L/Nix and PINK1/Parkin pathways. FUNDC1, FUN14 domain-containing 1; I/R, ischaemia/reperfusion.

Article Snippet: At the end of reperfusion, cells were fixed with 4% PFA and incubated with an antibody against FUNDC1 (1:100; CST, 49240).

Techniques:

Journal: Redox Biology

Article Title: Neutrophil extracellular traps drive intestinal microvascular endothelial ferroptosis by impairing Fundc1-dependent mitophagy

doi: 10.1016/j.redox.2023.102906

Figure Lengend Snippet: Effects of Fundc1 regulation on NET formation and endothelial mitochondrial quality control. A . Intestinal CitH3-DNA and MPO-DNA complexes were assayed using ELISAs. B–C . Protein expression levels of Ly6G and CitH3 were measured by Western blot analysis. D-E . Intestinal CD31-positive endothelial cells were isolated with CD31-coated magnetic beads. Mitochondrial (MitoTracker) and lysosomal (LysoTracker) immunofluorescence colocalization was performed to explore the mitophagy level. F-G . Endothelial mitophagy parameters (p62, LC3 II/I ratio and mito-LC3 II) and a mitochondrial protein (Tim23) were examined using western blotting. The relative grayscale values were measured using ImageJ. H–I . Cytc levels in the cytosol and isolated mitochondria were examined by western blotting. J-K . Quantification of cytoplasmic and mitochondrial ROS in the intestinal endothelium was conducted using DCFH‐DA and MitoSOX Red probes. L . Mitochondrial membrane potential changes in endothelial cells were detected by JC-1 staining. M-N . Key proteins of mitochondrial fusion (Mfn1 and Opa1) and fission (Mff and Drp1) were examined by a Western blot assay. O–P . Immunofluorescence confocal imaging of mitochondrial (Tom20, green) and Drp1 (red) colocalization was used to evaluate mitochondrial fission. AAV, adeno-associated virus; NC, negative control. Data are shown as the means ± SD, ns, not significant, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Rabbit antibodies against p Ser17 -Fundc1 and p Tyr18 -Fundc1 (1:500) were custom made by Abgent (Suzhou) according to a previous study [ , ].

Techniques: Expressing, Western Blot, Isolation, Magnetic Beads, Immunofluorescence, Membrane, Staining, Imaging, Virus, Negative Control

Journal: Redox Biology

Article Title: Neutrophil extracellular traps drive intestinal microvascular endothelial ferroptosis by impairing Fundc1-dependent mitophagy

doi: 10.1016/j.redox.2023.102906

Figure Lengend Snippet: Impact of Fundc1 regulation on the intestinal endothelial ferroptosis level and microvascular function. A . Endothelial cells isolated from intestinal tissue were used to evaluate MDA, GSH, GSSH and Fe 2+ levels by ELISAs. B–C . Intracellular Fe 2+ in the intestinal endothelium was evaluated using the FerroOrange probe. D-E . Western blot analysis was used to detect ferroptosis-related proteins in endothelial cells isolated from the intestine. F-G . Intestinal blood perfusion in WT and Fundc1 ΔEC mice was measured using a laser speckle blood flow analysis system. H . Intestinal and serum syndecan-1 levels were assessed using an ELISA kit. I-J . Pathological staining and immunohistochemistry were performed to evaluate intestinal microvascular function. The intestinal microvascular morphology was observed by HE and Masson’s trichrome staining and evaluated and graded according to the Chiu score system. TUNEL staining was used to evaluate microvascular cells undergoing apoptosis. Intestinal microvascular damage and permeability were assessed using immunohistochemistry for VCAM-1 and albumin. K-L . Western immunoblotting was used to examine VCAM-1 and VE-cadherin protein expression. M-N . To evaluate microvascular damage, immunofluorescence colocalization of CD31 and VE-cadherin was conducted in the intestine. Data correspond to the means ± SD, ns, not significant, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Rabbit antibodies against p Ser17 -Fundc1 and p Tyr18 -Fundc1 (1:500) were custom made by Abgent (Suzhou) according to a previous study [ , ].

Techniques: Isolation, Western Blot, Enzyme-linked Immunosorbent Assay, Staining, Immunohistochemistry, TUNEL Assay, Permeability, Expressing, Immunofluorescence

Journal: Redox Biology

Article Title: Neutrophil extracellular traps drive intestinal microvascular endothelial ferroptosis by impairing Fundc1-dependent mitophagy

doi: 10.1016/j.redox.2023.102906

Figure Lengend Snippet: During intestinal ischemia‒reperfusion, neutrophils become activated and infiltrate the intestinal microcirculation by releasing NETs. The presence of NETs in microvessels affects endothelial cells, resulting in the phosphorylation of Fundc1 at Tyr18, which inhibits mitophagy function. Consequently, damaged mitochondria are not cleared promptly, leading to mitochondrial dysfunction, which is characterized by the accumulation of mitochondrial ROS and by Cytc leakage. The accumulation of intracellular and mitochondrial ROS triggers lipid peroxidation, which activates ferroptosis in endothelial cells, ultimately causing microvascular damage and dysfunction. The clearance of NETs has been shown to effectively maintain mitophagy levels in endothelial cells, leading to enhanced mitochondrial quality control and a reduction in mitochondrial dysfunction. As a result, ROS generation is mitigated, cell ferroptosis is prevented, and intestinal microvascular function is preserved.

Article Snippet: Rabbit antibodies against p Ser17 -Fundc1 and p Tyr18 -Fundc1 (1:500) were custom made by Abgent (Suzhou) according to a previous study [ , ].

Techniques: