Journal: The Journal of Clinical Investigation

Article Title: Inhibition of mitophagy drives macrophage activation and antibacterial defense during sepsis

doi: 10.1172/JCI130996

Figure Lengend Snippet: (A–D) Flow cytometry assessment of mitochondrial ROS (upper) and mitochondrial density (lower) in raw 264.7 macrophages (A) exposed to TLR agonists, (B) mitophagy inhibitor (mdivi-1), or (C and D) to LPS and IFN-γ alone or in combination for 24 hours or a specified duration (n = 3 per condition). (E) Flow cytometry assessment of mitophagy in raw 264.7 macrophages with mt-mkeima (upper) or autophagic vacuole formation with CYTO-ID autophagy detection kit (lower) in raw 264.7 macrophages exposed to LPS/IFN-γ (n = 3 per condition). (F and G) Immunoblots of mitophagy and mitochondrial fission checkpoints on protein lysates from (F) stat1+/+ or stat1–/– BMDMs exposed to LPS/IFN-γ or (G) from raw 264.7 macrophages exposed for 24 hours to LPS/IFN-γ alone or in combination with a mitophagy inducer (2,4-DNP, 1 μM) (densitometry: ratio to β-actin is presented above the immunoblots). Bar graphs represent mean ± SEM with overlaid individual values; #P < 0.05, ##P < 0.01, ###P < 0.001 determined by ANOVA corrected for multiple comparisons; *P < 0.05, ***P < 0.001 determined by Student’s t test with Welch’s correction.

Article Snippet: Mdivi-1 (Bio-Techne) was diluted in sterile saline (1% DMSO) and gently sonicated before i.p. injection (50 mg/kg) to obtain a homogenous suspension.

Techniques: Flow Cytometry, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: Inhibition of mitophagy drives macrophage activation and antibacterial defense during sepsis

doi: 10.1172/JCI130996

Figure Lengend Snippet: (A and B) Mitochondrial network of (A) mouse peritoneal macrophages or (B) raw 264.7 macrophages exposed to vehicle or LPS/IFN-γ for 6 hours (A) immunostained for Tom20 in combination or not with PINK1 or (B) stained with JC-1 (right panels represent the image binarization of JC-1 stained macrophages). Scale bars: 10 μm. (C) Flow cytometry assessment of mitochondrial density and Δψm in raw 264.7 macrophages exposed to LPS/IFN-γ and stained with JC-1 (n = 3 per time point). (D and E) (D) Oxygen consumption (OCR) and extracellular acidification (ECAR) profile measured with Seahorse XFe96 analyzer on BMDMs exposed to vehicle, LPS/IFN-γ for 6 hours, or mdivi-1 for 24 hours (n = 8 per condition). (F) Flow cytometry assessment of Δψm with TMRM in raw 264.7 macrophages exposed to LPS/IFN-γ for 6 hours (n = 3 per condition). (G) Cell death assessed by low cytometry (annexin V-PI) in raw 264.7 macrophages exposed to vehicle or LPS/IFN-γ and 2,4 DNP alone or in combination for 18 hours. Bar graphs represent mean ± SEM with overlaid individual values; #P < 0.05, ##P < 0.01, ###P < 0.001 determined by ANOVA corrected for multiple comparisons; **P < 0.01, ***P < 0.001 determined by Student’s t test with Welch’s correction. Veh, vehicle.

Article Snippet: Mdivi-1 (Bio-Techne) was diluted in sterile saline (1% DMSO) and gently sonicated before i.p. injection (50 mg/kg) to obtain a homogenous suspension.

Techniques: Staining, Flow Cytometry, Cytometry

Journal: The Journal of Clinical Investigation

Article Title: Inhibition of mitophagy drives macrophage activation and antibacterial defense during sepsis

doi: 10.1172/JCI130996

Figure Lengend Snippet: (A) Flow cytometry assessment of classical macrophage activation in raw 264.7 cells exposed to antimycin A for 6 hours. (B and C) Flow cytometry assessment of classical macrophage activation in raw 264.7 cells exposed to LPS/IFN-γ for 18 hours alone or in combination with (B) the mitochondrial ROS scavenger MitoTEMPOL or with (C) the HIF-1α inhibitor echinomycin (n = 3 per condition). (D–I) Assessment of macrophage activation profile by (D and G) flow cytometry, (E and H) gene expression, and (F and I) bactericidal activity in raw 264.7 cells (D, E, G, and H) incubated (24 hours) or (F and I) preincubated (24 hours) with (D–F) the mitophagy inhibitor mdivi-1 or (G–I) the mitophagy inducer CCCP (n = 3 per condition). Bar graphs represent mean ± SEM with overlaid individual values; #P < 0.05, ##P < 0.01, ###P < 0.001 determined by ANOVA corrected for multiple comparisons; *P < 0.05, ***P < 0.001 determined by Student’s t test with Welch’s correction.

Article Snippet: Mdivi-1 (Bio-Techne) was diluted in sterile saline (1% DMSO) and gently sonicated before i.p. injection (50 mg/kg) to obtain a homogenous suspension.

Techniques: Flow Cytometry, Activation Assay, Expressing, Activity Assay, Incubation

Journal: The Journal of Clinical Investigation

Article Title: Inhibition of mitophagy drives macrophage activation and antibacterial defense during sepsis

doi: 10.1172/JCI130996

Figure Lengend Snippet: (A) Survival curve of C57BL6/J mice treated with saline (sal.) or 2,4-DNP (10 mg/kg) 24 hours before sham or CLP surgery (sham, n = 5 per group; CLP, n = 13 per group). P value was determined by Gehan-Breslow-Wilcoxon test. (B) Percentage of classically activated macrophages (percentage of CD11bhi F4/80hi macrophages) in C57BL6/J mice treated as in A (n = 7 per condition). (C) Bacterial load in the peritoneal cavity of C57BL6/J mice treated as in A (n = 8 per condition). (D) Correlation of the mitochondrial density in Ly6Chi blood monocytes 2 hours after CLP surgery versus the survival (in hours) after CLP surgery (n = 26). P and r values were determined by Spearman’s rank correlation. (E) Survival curve of C57BL6/J mice treated with vehicle or mdivi-1 for 24 hours before CLP surgery (CLP + vehicle, n = 15; CLP + mdivi-1, n = 15). P value was determined by Gehan-Breslow-Wilcoxon test. (F and G) Percentage of classically activated macrophages (F) (percentage of CD11bhi F4/80hi macrophages) and bacterial load (G) in the peritoneal cavity of C57BL6/J mice treated as in E (n = 6–7 per condition) (H) Survival curve of C57BL6/J mice transplanted with Pink1+/+ (BMT Pink1+/+) or Pink1–/– bone marrow (BMT Pink1–/–) 5 weeks before CLP surgery (BMT Pink1+/+, n = 13; BMT Pink1–/–, n = 15). P value was determined by Gehan-Breslow-Wilcoxon test. (I) Flow cytometry assessment of the percentage of classically activated macrophages (percentage of CD11bhi F4/80hi macrophages) in the peritoneal cavity of mice that underwent transplantation and surgery as in H (n = 11–12 per group). (J) Bacterial load in the cavity of mice that underwent transplantation and surgery as in H (n = 11–12 per group). Graphs with plots represent mean plus individual values; *P < 0.05 determined by Student’s t test with Welch’s correction.

Article Snippet: Mdivi-1 (Bio-Techne) was diluted in sterile saline (1% DMSO) and gently sonicated before i.p. injection (50 mg/kg) to obtain a homogenous suspension.

Techniques: Flow Cytometry, Transplantation Assay

Journal: Frontiers in Pharmacology

Article Title: Evaluation of the efficacy of mitochondrial fission inhibitor (Mdivi-1) using non-alcoholic steatohepatitis (NASH) liver organoids

doi: 10.3389/fphar.2023.1243258

Figure Lengend Snippet: Structures and functions of mitochondria in non-alcoholic steatohepatitis (NASH) liver organoids. To generate and analyze NASH liver organoids (NLO), liver tissues were harvested from NASH model mice induced by feeding a high-fat diet, not including methionine and choline for 12 weeks (A) . Bright-field (BF) and transmission electron microscopy (TEM) images of control and NASH liver organoids. BF: Scale bar: 100 μm, TEM: Scale bar: 600 nm (B) . Arrows show the typical dendritic-like cells (B) . Schematic diagram of the relationship between mitochondrial fission/fusion and reactive oxygen species (ROS) in NASH liver tissues. Glucose and oxygen stimulate mitochondria-derived ROS production, which induces cytotoxicity of the hepatocyte. Mdivi-1 inhibits mitochondrial division factor (DRP1), while MYLS22 inhibits mitochondrial fusion factor (OPA1) (C) . Mitochondria-derived ROS production in NLO. Representative images for mtSOX Deep Red staining of control and NASH liver organoids (CLO and NLO). Scale bar: 100 μm (D) , (n = 3). Fluorescence intensity in the stained images was quantified by using ImageJ software. Results were shown as fold increase relative to CLO and expressed as mean ± S.E.M. * p < 0.05 vs. CLO (E) . Protein expression level of DRP1, MFF, and OPA1 was compared between CLO and NLO as determined by Western blotting. Equal loading of protein was confirmed by using a total Valosin-containing protein (VCP) antibody. Quantification of protein expression level was analyzed by ImageJ software (E, n = 3–5). Results are expressed as mean ± S.E.M. * p < 0.05 vs. CLO.

Article Snippet: DRP1 inhibitor (Mdivi-1, M3108; Tokyo Kasei Kogyo Co., Ltd., Tokyo) and OPA1 inhibitor (MYLS22, S9885; Selleck chem.com , USA) were obtained commercially.

Techniques: Transmission Assay, Electron Microscopy, Control, Derivative Assay, Staining, Fluorescence, Software, Expressing, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Evaluation of the efficacy of mitochondrial fission inhibitor (Mdivi-1) using non-alcoholic steatohepatitis (NASH) liver organoids

doi: 10.3389/fphar.2023.1243258

Figure Lengend Snippet: Summary of the effects of Mdivi-1 on NASH progression. In NASH pathology, overexpression of DRP1 in hepatocytes triggers aberrant mitochondrial dynamics, increases ROS production, fat accumulation, and fibrosis. Treatment of NASH organoids or NASH mice with Mdivi-1 reduced ROS production, which might lead to decreased lipid accumulation and fibrosis in the NASH progression.

Article Snippet: DRP1 inhibitor (Mdivi-1, M3108; Tokyo Kasei Kogyo Co., Ltd., Tokyo) and OPA1 inhibitor (MYLS22, S9885; Selleck chem.com , USA) were obtained commercially.

Techniques: Over Expression