Journal: Redox Biology

Article Title: STING signaling sensing of DRP1-dependent mtDNA release in kupffer cells contributes to lipopolysaccharide-induced liver injury in mice

doi: 10.1016/j.redox.2022.102367

Figure Lengend Snippet: DRP1-mediated mitochondrial fission triggers STING signaling activation in LPS-treated KCs. (A–B) Western blot analysis of DRP1 protein expression in KCs isolated from WT mice after Control or LPS treatment (n = 6). (C) Representative of immunofluorescence staining of MitoTracker and p-DRP1 in KCs (bar = 5 μm). (D–F) Mitochondrial network parameters including mitochondrial length, mitochondrial branch junctions and the percentage of mitochondrial with different lengths in KCs transfected with negative control vector (NC) or shDRP1 in the absence or presence of LPS (100 ng/mL for 12 h, n = 40 cells). (G) Co-location analysis of mitochondrial and p-DRP1 in each group (n = 12 cells). (H–K) Western blot analysis of STING, IRF3, p-IRF3, p65 and p-p65 protein expression in KCs transfected with NC or shDRP1 in the absence or presence of LPS (n = 4). Data are expressed as mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The primary antibodies used for immunofluorescence were anti -p-DRP1 (1:300, Cell signaling, #3455) and anti -DNA (1:100, Progen, AC-30-10), respectively.

Techniques: Activation Assay, Western Blot, Expressing, Isolation, Control, Immunofluorescence, Staining, Transfection, Negative Control, Plasmid Preparation

Journal: Redox Biology

Article Title: STING signaling sensing of DRP1-dependent mtDNA release in kupffer cells contributes to lipopolysaccharide-induced liver injury in mice

doi: 10.1016/j.redox.2022.102367

Figure Lengend Snippet: Mitochondrial fission-induced mtDNA release mediates STING signaling activation in LPS-treated KCs. (A) Representative of immunofluorescence staining of Mitotracker and dsDNA in KCs (bar = 5 μm). (B) Relative numbers of cytosolic dsDNA in KCs transfected with NC or shDRP1 in the absence or presence of LPS (n = 15 cells). (C) Cytosolic mtDNA fragments of D-loops levels in KCs transfected with NC or shDRP1 in the absence or presence of LPS (n = 3). (D–F) Cytosolic mtDNA D-loops levels in KCs transfected with empty vector (EV) or GV-314 vector for over-expressing DRP1 followed by incubation of DNase I or heat-inactivated DNase I (HI DNaseI) and then treated with LPS (n = 3). (G) IFN-β mRNA levels in KCs treated as in D (n = 3). Data are expressed as mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The primary antibodies used for immunofluorescence were anti -p-DRP1 (1:300, Cell signaling, #3455) and anti -DNA (1:100, Progen, AC-30-10), respectively.

Techniques: Activation Assay, Immunofluorescence, Staining, Transfection, Plasmid Preparation, Expressing, Incubation

Journal: Redox Biology

Article Title: STING signaling sensing of DRP1-dependent mtDNA release in kupffer cells contributes to lipopolysaccharide-induced liver injury in mice

doi: 10.1016/j.redox.2022.102367

Figure Lengend Snippet: The oxidative stress induced by DRP1-dependent mitochondrial fission enhances the release of mtDNA and subsequent STING signaling activation in LPS-treated KCs. (A–B) Representative of immunofluorescence staining of MitoSOX in KCs transfected with NC or shDRP1 in the absence or presence of LPS (n = 12 cells, bar = 5 μm). (C) Cytosolic mtDNA D-loops levels in KCs pre-treated with DMSO or MitoQ and then stimulated with LPS (n = 3). (D) IFN-β mRNA levels in KCs treated as in C (n = 3). Data are expressed as mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The primary antibodies used for immunofluorescence were anti -p-DRP1 (1:300, Cell signaling, #3455) and anti -DNA (1:100, Progen, AC-30-10), respectively.

Techniques: Activation Assay, Immunofluorescence, Staining, Transfection

Journal: Redox Biology

Article Title: STING signaling sensing of DRP1-dependent mtDNA release in kupffer cells contributes to lipopolysaccharide-induced liver injury in mice

doi: 10.1016/j.redox.2022.102367

Figure Lengend Snippet: Inhibition of mitochondrial fission with Mdivi-1 attenuates LPS-induced STING signaling activation in KCs and protects liver function. Primary hepatocytes and KCs were isolated from WT mice treated with Mdivi-1 20 mg/kg or Vehicle (equal volume of DMSO) in the absence or presence of LPS (10 mg/kg for 12 h). (A–E) Western blot analysis of STING, IRF3, p-IRF3, p65, p-p65, DRP1, p-DRP1 protein expression in KCs (n = 6). (F–H) Levels of inflammatory cytokines including IFN-β, TNF-α and IL-1β in mouse serum in each group (n = 4). (I–J) Hepatocyte death detection by flow cytometry in each group (n = 4). (K–L) H&E staining and histology scores of liver sections in each group (n = 6, bar = 100 μm). (M − N) ALT and AST concentrations in mouse serum in each group (n = 6). Data are expressed as mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The primary antibodies used for immunofluorescence were anti -p-DRP1 (1:300, Cell signaling, #3455) and anti -DNA (1:100, Progen, AC-30-10), respectively.

Techniques: Inhibition, Activation Assay, Isolation, Western Blot, Expressing, Flow Cytometry, Staining

Journal: Redox Biology

Article Title: STING signaling sensing of DRP1-dependent mtDNA release in kupffer cells contributes to lipopolysaccharide-induced liver injury in mice

doi: 10.1016/j.redox.2022.102367

Figure Lengend Snippet: Schematic process of the activation of STING signaling mediated by DRP1-dependent release of mtDNA in KCs in LPS-induced liver injury.

Article Snippet: The primary antibodies used for immunofluorescence were anti -p-DRP1 (1:300, Cell signaling, #3455) and anti -DNA (1:100, Progen, AC-30-10), respectively.

Techniques: Activation Assay

Journal: Journal of Nanobiotechnology

Article Title: Young small extracellular vesicles rejuvenate replicative senescence by remodeling Drp1 translocation-mediated mitochondrial dynamics

doi: 10.1186/s12951-024-02818-5

Figure Lengend Snippet: Impaired Drp1-mediated mitochondrial dynamism induces a senescent-like phenomenon in BMSCs. a Colocalization analysis of Drp1 (green) and mitochondria (red). Scale bar, 10 μm. MOC were analyzed by Zeiss LSM 980. b RT-qPCR assay of Drp1. c Representative images of SA-β-gal positive BMSCs. Scale bar, 50 μm. d Quantification of SA-β-gal positive BMSCs. e Representative images of EdU-positive BMSCs. Scale bar, 50 μm. f Quantification of EdU-positive BMSCs. g Representative images of ALP staining of BMSCs treated as indicated after osteogenic induction for 7 days. Data is shown as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ns: not significant ( p > 0.05)

Article Snippet: After fixation and blocking, cells were incubated with rabbit anti-human Drp1 primary antibody (1:100, BOSTER) in a wet box overnight at 4 °C.

Techniques: Quantitative RT-PCR, Staining

Journal: Journal of Nanobiotechnology

Article Title: Young small extracellular vesicles rejuvenate replicative senescence by remodeling Drp1 translocation-mediated mitochondrial dynamics

doi: 10.1186/s12951-024-02818-5

Figure Lengend Snippet: Young sEVs rejuvenate replicative senescent BMSCs by promoting Drp1 translocation onto mitochondria. a SER texture analysis in BMSCs. Scale bar, 50 μm. b Analysis of mitochondrial membrane potential (MMP) using JC-1 staining. Quantification of depolarized cells was calculated. c Quantification of ATP levels per group. d Quantification of the intercellular ROS levels using mean fluorescence intensity of the DCFH-DA. e Quantification of the mitoSOX-positive mitochondria in all mitochondria. f Representative images of western blot analysis showing the expression levels of Drp1 and β-actin. g Representative images of colocalization of mitochondria (red), and Drp1 (green) per group. Scale bar, 10 μm. h Representative images of western blot analysis showing the change of mitochondrial fission gene Drp1. Data is shown as mean ± SEM. Comparison with P3 BMSCs: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ns: no significant difference. Comparisons between identical passage: # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001. NS: no significant difference

Article Snippet: After fixation and blocking, cells were incubated with rabbit anti-human Drp1 primary antibody (1:100, BOSTER) in a wet box overnight at 4 °C.

Techniques: Translocation Assay, Membrane, Staining, Fluorescence, Western Blot, Expressing, Comparison

Journal: Redox Biology

Article Title: FBXL4 protects against HFpEF through Drp1-Mediated regulation of mitochondrial dynamics and the downstream SERCA2a

doi: 10.1016/j.redox.2024.103081

Figure Lengend Snippet: Adult mice were fed HFD and l -NAME in drinking water for 15 weeks prior to TMT-proteomic analysis of heart tissues. A: Cellular components (CC); B: Biological process (BP); C: Chord diagram displaying the interplay of protein and KEGG pathways; D: Heat map exhibiting up- or down-regulated genes; E: Levels of F-box and leucine-rich repeat protein 4 (FBXL4); F: Levels of mitochondrial fission proteins dynamin-related protein 1 (Drp1), mitochondrial fission protein 1 (FIS1) and mitofusin-1 (MFF); and G: Levels of mitochondrial fusion proteins mitofusin-1 (OPA1), MFN1 and MFN2; Insets: Representative gel bands depicting FBXL4 and mitochondrial fusion/fission proteins using specific antibodies; Mean ± SEM, n = 3 mice for TMT proteomics (panel A–D) or 6 mice per group (panel E–G). Differential proteins were identified using variable important in projection (VIP) > 1.2 in an orthogonal partial least square-discriminant analysis (OPLS-DA) model. Differentially expressed genes were defined as genes with a Benjamini-Hochberg adjusted P value < 0.05. Statistical significance was estimated using one-way ANOVA, *** p < 0.001, **** p < 0.0001 between indicated groups.

Article Snippet: After tissue or cell lysates were centrifuged at 22,500× g at 4 °C for 15 min, supernatants were incubated with anti-Drp1 beads (Shanghai Genomics Inc., Shanghai, China) at 4 °C overnight.

Techniques:

Journal: Redox Biology

Article Title: FBXL4 protects against HFpEF through Drp1-Mediated regulation of mitochondrial dynamics and the downstream SERCA2a

doi: 10.1016/j.redox.2024.103081

Figure Lengend Snippet: Effect of FBXL4 on HFpEF-induced changes in mitochondrial dynamics and Ca 2+ transport. A: Representative immunoblots depicting pan and phosphorylated (Ser 616/637 ) Drp1; B: Drp1; C–D: Phosphorylated Drp1 ( p -Drp1 Ser 616 , Ser 637 ); E: p -Drp1 Ser 616 -to-Drp1 ratio; F: Representative immunoblots depicting FIS1 and MFF using specific antibodies; G: FIS1; H: MFF; I: Representative immunoblots of OPA1, MFN1 and MFN2 using specific antibodies; J: OPA1; K: MFN1; L: MFN2; M: Representative immunoblots of SERCA2a, PLN and NCX using specific antibodies; N: Sarcoplasmic/endoplasmic reticulum Ca 2+ -ATPase 2a (SERCA2a); O: Phospholamban (PLN); P: Na + -Ca 2+ exchanger (NCX); Q: Representative immunoblots of inositol 1,4,5-trisphosphate receptor 1 (IP3R1), IP3R3, and ryanodine receptor 1 (RYR1) using specific antibodies; R: IP3R1; S: IP3R3; and T: RYR1. Mean ± SEM, n = 10 mice per group (panel A–E), n = 6 mice per group (panel F–T). Statistical significance was estimated by one-way ANOVA followed by Tukey's multiple comparison test. * p < 0.05, *** p < 0.001, **** p < 0.0001 between indicated groups.

Article Snippet: After tissue or cell lysates were centrifuged at 22,500× g at 4 °C for 15 min, supernatants were incubated with anti-Drp1 beads (Shanghai Genomics Inc., Shanghai, China) at 4 °C overnight.

Techniques: Western Blot, Comparison

Journal: Redox Biology

Article Title: FBXL4 protects against HFpEF through Drp1-Mediated regulation of mitochondrial dynamics and the downstream SERCA2a

doi: 10.1016/j.redox.2024.103081

Figure Lengend Snippet: Metabolomics and proteomics analyses in plasma from HFpEF patients and mouse hearts, as well as establishment of an in vitro HFpEF model for diastolic dysfunction. A: Metabolomics analysis of human plasma (male only) using Mass spectrometry; B: Metabolomics analysis of mouse heart tissues; C: KEGG pathways of top 10 differential proteins from HFpEF mouse hearts using TMT-proteomics; D: Protocol depicting the in vitro ‘double-damage’ (DD) model established using high glucose (HG, 12.5 mM) and palmitic acid (PA, 0.2 mM) in cardiomyocytes from WT, Drp1 CKI and Drp1 hetCKO mice; E: Peak shortening (% cell length); F: Maximal velocity of shortening (+dL/dt); G: Maximal velocity of relengthening (-dL/dt); H: Time-to-10%, 50% and 90% shortening (TP10, TP50 and TP90); I: Time-to-10%, 50%, and 90% relengthening (TR10, TR50 and TR90); J: Representative tetramethylrhodamine, methyl ester (TMRM) staining of mitochondrial membrane potential (red color) using confocal microscopy; K: Pooled data of mitochondrial membrane potential using TMRM; L–P: Adult mouse cardiomyocytes from Drp1 CKI and Drp1 hetCKO mice were transfected with Adv-FBXL4 or vector for 48 h prior to ‘double-damage’ challenge for another 4 h. L: Peak shortening; M: + dL/dt; N: dL/dt; O: Time-to-90% shortening (TP90); and P: Time-to-90% relengthening (TR90). Mean ± SEM, Independent-samples used t -test or Wilcoxon rank sum test for continuous variables and Chi-square test for categorical variables (details provided in ); n = 15 subjects (panel A) for metabolomics per group; n = 6 mice (panel B) for metabolomics; n = 3 mice (panel C) for TMT-proteomics, Differential proteins were identified using variable important in projection (VIP) values of >1.2 in an orthogonal partial least square–discriminant analysis (OPLS-DA) model. n = 6 mice per group (10 replicates per heart, panel E–I), and n = 6 mice per group (5 replicates per heart, panel J–P). Statistical significance was estimated using one-way ANOVA followed by the Tukey's multiple comparison test, repeated measures ANOVA in R and Bonferroni adjusted test. *** p < 0.001, **** p < 0.0001 between indicated groups.

Article Snippet: After tissue or cell lysates were centrifuged at 22,500× g at 4 °C for 15 min, supernatants were incubated with anti-Drp1 beads (Shanghai Genomics Inc., Shanghai, China) at 4 °C overnight.

Techniques: In Vitro, Mass Spectrometry, Staining, Membrane, Confocal Microscopy, Transfection, Plasmid Preparation, Comparison

Journal: Redox Biology

Article Title: FBXL4 protects against HFpEF through Drp1-Mediated regulation of mitochondrial dynamics and the downstream SERCA2a

doi: 10.1016/j.redox.2024.103081

Figure Lengend Snippet: Detection of Drp1-FBXL4 binding and FBXL4-Drp1 axis in ‘double-damage’-evoked mitochondrial fragmentation. A–B: Structure-based interface analysis between Drp1 and FBXL4. A: FBXL4-Drp1 complex structure with interaction hot-spot residues labeled using PRISM ; B: Domain deletion of FBXL4 for Co-IP experiments, and predicted Drp1 interaction sites on FBXL4. Boxes indicate exons; white bars indicate deleted domains; C: IP analysis of FBXL4 tagged with Flag (FBXL4-Flag) and Drp1 tagged with His (Drp1-His) in AMCM cells from chow and HFpEF; D: IP analysis of FBXL4 domain mutations (FBXL4-Flag, FBXL4-ΔFbox, FBXL4-ΔLRR2) using Drp1-His in H9C2 cell line; E: H9C2 cells were divided into 4 groups (FBXL4-Flag, FBXL4-ΔFbox, FBXL4-ΔLRR2, Fbox) prior to transfection of Drp1 using plasmid DNA, Confocal image exhibiting H9C2 cells transfected with indicated plasmids, stained with MitoTracker (red), anti-FBXL4 (green), and anti-Drp1 (blue) antibodies; F–G: Colocalization and Pearson's correction coefficient; H–I: Assessment of mitochondrial structure using confocal microscopy in adult mouse cardiomyocytes (AMCM) transfected with Adv-FBXL4 for 48 h prior to DD challenge for 12 h; H: Representative mitochondrial immunofluorescence using Mito Tracker (red); and I: Quantification of mitochondrial length of Drp1 CKI and Drp1 hetCKO groups. J–M: Ubiquitylation level of Drp1 in heart tissues from chow and HFpEF with or without FBXL4 overexpression; N–P: Drp1 level in H9C2 cells transfected with FBXL4-siRNA or FBXL4-scramble, treated with or without MG132 for 4, 8, and 12 h; Q–S: Whole cell level of Drp1 treated with or without MG132 for 4, 8, and 12 h in AMCM cells from chow and HFpEF; Mean ± SEM, n = 3 mice per group (5 replicates per heart, panel E–I), or 3 replicates from 1 sample per group (panel J–M), or 2 replicates from 3 sample per group (panel N–S). Statistical significance was estimated by one-way ANOVA followed by the Tukey's multiple comparison test or repeated measures ANOVA in R and Bonferroni adjusted test. * p < 0.05, *** p < 0.001, **** p < 0.0001 between indicated groups.

Article Snippet: After tissue or cell lysates were centrifuged at 22,500× g at 4 °C for 15 min, supernatants were incubated with anti-Drp1 beads (Shanghai Genomics Inc., Shanghai, China) at 4 °C overnight.

Techniques: Binding Assay, Labeling, Co-Immunoprecipitation Assay, Transfection, Plasmid Preparation, Staining, Confocal Microscopy, Immunofluorescence, Over Expression, Comparison

Journal: Redox Biology

Article Title: FBXL4 protects against HFpEF through Drp1-Mediated regulation of mitochondrial dynamics and the downstream SERCA2a

doi: 10.1016/j.redox.2024.103081

Figure Lengend Snippet: Role of ATP-dependent SERCA2a/PLN and Drp1 in FBXL4-offered benefits in ‘double-damage’ cell model. A–F: Experiments depicting intracellular Ca 2+ transient properties in control and ‘double damage’ (DD) mouse cardiomyocytes; G–J: Caffeine-induced changes in baseline fura-2 fluorescence intensity (FFI), ΔFFI and intracellular Ca 2+ decay rate; K–M: Impact of SERCA inhibition using thapsigargin (Tg) on FBXL4-offered benefit against DD-induced intracellular Ca 2+ mishandling including baseline Fura-2 fluorescent intensity; N–P: Effect of FBXL4 on DD-induced myocardial energy deficit using OCR curve (panel N), basal OCR and maximal respiration (panel O–P); and Q–S: Effect of Drp1 knock-in or knock-out in FBXL4-offered intracellular Ca 2+ responses under DD challenge. Cardiomyocytes from WT, Drp1 CKI and Drp1 hetCKO mice were challenged with DD regimen for 12 h in the absence or presence of FBXL4 overexpression; Mean ± SEM, n = 6 mice per group (5 replicates per heart, panel B–F, K–S), n = 5 mice per group (3 replicates per heart, panel G–J). Statistical significance was estimated using one-way ANOVA followed by the Tukey's multiple comparison test or repeated measures ANOVA in R and Bonferroni adjusted test. *** p < 0.001, **** p < 0.0001 between indicated groups.

Article Snippet: After tissue or cell lysates were centrifuged at 22,500× g at 4 °C for 15 min, supernatants were incubated with anti-Drp1 beads (Shanghai Genomics Inc., Shanghai, China) at 4 °C overnight.

Techniques: Fluorescence, Inhibition, Knock-In, Knock-Out, Over Expression, Comparison

Journal: Redox Biology

Article Title: FBXL4 protects against HFpEF through Drp1-Mediated regulation of mitochondrial dynamics and the downstream SERCA2a

doi: 10.1016/j.redox.2024.103081

Figure Lengend Snippet: Schematic diagram exhibiting possible involvement of FBXL4 in HFpEF-associated cardiomyopathy through FBXL4-evoked Drp1 degradation in a proteasomal-dependent manner. HFpEF downregulates FBXL4 level, resulting hyperactivation of Drp1 and subsequent mitochondrial injury. Elevated FBXL4 level rescues against HFpEF-induced cardiac remodeling, diastolic dysfunction, and mitochondrial injury (mitochondrial fragmentation, collapsed membrane potential and intracellular Ca 2+ overload due to defective SERCA2a function) through reverting hyperactivation of Drp1-mediated mitochondrial fission.

Article Snippet: After tissue or cell lysates were centrifuged at 22,500× g at 4 °C for 15 min, supernatants were incubated with anti-Drp1 beads (Shanghai Genomics Inc., Shanghai, China) at 4 °C overnight.

Techniques: Membrane