mito ros scavenger mito tempo  (Tocris)

Journal: Science Advances

Article Title: Mitochondrial ROS drive foam cell formation via STAT5 signaling in atherosclerosis

doi: 10.1126/sciadv.adw9952

Figure Lengend Snippet: ( A ) pYSTAT5 levels in naïve (M0) and LPS + IFN-γ–polarized hMDMs with or without DMNQ, assessed by flow cytometry. n = 7. ( B ) pYSTAT5 quantification in oxLDL-treated hMDMs (24 hours) ± S5i or mTEMPO pretreatment (both 1 hour). n = 4. ( C ) Mitochondrial superoxide (O 2 •− ) production in oxLDL-treated hMDMs (24 hours) ± S5i, determined by flow cytometry. n = 5. ( D ) Mitochondrial membrane potential (ΔΨ m ) assessment by flow cytometry in oxLDL-treated hMDMs (24 hours) ± S5i, mTEMPO (both 1 hour), or MitoQ (2 hours) pretreatment. Cells stained with TMRM and MTG and data shown as a ratio of TMRM + cells to MTG + cells (%). n = 5. ( E ) CD36 expression and representative flow cytometry histogram in hMDMs treated as in (B). n = 4. ( F ) Oil Red O staining in hMDMs treated as in (C). Lipids in red; nuclei in blue. The right panel shows a representative image and a zoomed-in inset. Arrows indicate lipid-positive cells. Scale bar, 50 μm; inset, 20 μm. n = 5. ( G ) Migration assay in oxLDL-treated mouse BMDMs from Stat5 -deficient mice [ Vav1 -Cre/+ Stat5ab fl /fl ( Stat5 −/− ) mice] and their wild-type counterpart ( Stat5 fl/fl ) ± mTEMPO or both. Data represent the migrated area, normalized to nonmigrated cells (%). n = 5. ( H ) Phagocytosis of pHrodo-labeled Escherichia coli particles in BMDMs treated as in (F), shown as % positive cells normalized to unstimulated controls, with representative histograms. n = 4. All data represent the means ± SD of independent biological replicates. (A) * P < 0.05, paired t test; [(B) to (F)] * P < 0.05, ** P < 0.01, one-way ANOVA with Tukey’s multiple comparison test; [(G) and (H)] * P < 0.05, ** P < 0.01, *** P < 0.001, two-way ANOVA with Tukey’s test (intragroup) and Sidak’s test (intergroup).

Article Snippet: For inhibition of mitoROS, the ROS scavenger mTEMPO (cat. no. sc-221945; Santa Cruz Biotechnology) was used with 1-hour pretreatment at the concentrations of 500 μM in hMDMs and 300 μM in BMDMs, and the mitochondrion-targeted antioxidant MitoQ (cat. no. S8978; Selleckchem) was used with 2-hour pretreatment at the concentration of 500 nM. oxLDL (cat. no. L34357 ; Thermo Fisher Scientific) was used at the concentration of 50 μg/ml in both cell types and DiI-oxLDL (cat. no. L34358; Thermo Fisher Scientific) at 50 μg/ml in hMDMs, both for 24 hours of stimulation. oxLDL and DiI-oxLDL stimulation was carried out in complete medium depleted of 10% FBS with the addition of 0.2% bovine serum albumin (BSA; Sigma-Aldrich).

Techniques: Flow Cytometry, Membrane, Staining, Expressing, Migration, Labeling, Comparison

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Mitochondrial oxidative stress inhibited Sirt3/Foxo3/PPARα pathway and aggravated copper and zinc co-deficiency-induced hepatic lipotoxicity in a fish model

doi: 10.1007/s00018-025-05756-z

Figure Lengend Snippet: Cu/Zn-SOD induced mtROS mediated the changes of Cu- and Zn- co-deficiency-induced mitochondrial damage, Sirt3, Foxo3, and PPARα expression in yellow catfish hepatocytes. Isolated yellow catfish hepatocytes were pre-treated with SOD analogue (3 mM Tempol) or mitochondrial ROS scavenger (15µM MitoTEMPO) 2 h in advance and then incubated in the control, Cu and Zn chelators containing medium for 48 h. ( A ) Relative quantification of Cu/Zn-SOD protein expression. ( B ) Western blot analysis of Cu/Zn-SOD protein expression. ( C ) Representative confocal microscopy image of hepatocytes stained with DHE. Scale bars, 25 μm. ( D ) Intracellular O 2 • − was quantified by calculating FL1 (red) mean fluorescence intensity (DHE fluorescent staining). ( E ) Fluorescence intensity of O 2 • − was measured by flow cytometry. ( F ) MDA content. ( G ) Intracellular mtROS was quantified by calculating FL1 (red) mean fluorescence intensity (MitoSOX fluorescent staining). ( H ) Fluorescence intensity of mtROS was measured by flow cytometry. ( I ) Representative confocal microscopy image of hepatocytes stained with MitoSOX. Scale bars, 7.5 μm. ( J ) TG content. ( K ) Representative confocal microscopic image of hepatocytes stained with JC-1. Red fluorescence indicates normal ΔΨm with JC-1 aggregates in mitochondria and green fluorescence reflects cytosolic JC-1 monomer indicative of ΔΨm loss. Scale bar, 10 μm. ( L ) The MMP was quantified by flow cytometric analysis of red/green (FL2/FL1) fluorescence ratio (JC-1 fluorescent staining, 5µM). ( M ) The presence of JC-1 intracellular mitochondrial transmembrane potential (MMP) was determined by flow cytometry analysis of red/green (FL2/FL1) fluorescence ratio. ( N ) Sirt3, T-Foxo3, N-Foxo3 and PPARα protein levels were detected by Western blotting. ( O ) Relative quantification of Sirt3, T-Foxo3, N-Foxo3 and PPARα protein expression. ( P ) TG content. All data are expressed as mean ± S.E.M. ( n = 3 independently biological experiments). P value was calculated by Student’s t tests. Asterisk (*) indicate significant difference between two groups ( P < 0.05). CuD: Cu deficiency; ZnD: Zn deficiency

Article Snippet: To explore mechanisms of oxidative stress and the Sirt3 pathway in Cu- and Zn- deficiency-induced lipid deposition, we utilized the superoxide dismutase analog Tempol (3 mM, MedChem Express, NJ, USA, HY-100561), the mitochondrial ROS scavenger MitoTEMPO (15μM, MedChem Express, HY-112879) and Honokiol (HNK, MedChem Express, HY-N0003) in experiments with cultured yellow catfish hepatocytes.

Techniques: Expressing, Isolation, Incubation, Control, Quantitative Proteomics, Western Blot, Confocal Microscopy, Staining, Fluorescence, Flow Cytometry

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Impeding Nucleotide‐Binding Oligomerization Domain‐Like Receptor 3 Inflammasome Ameliorates Cardiac Remodeling and Dysfunction in Obesity‐Associated Cardiomyopathy

doi: 10.1161/JAHA.124.035234

Figure Lengend Snippet: A through H , NRVMs treated with PBS or MitoTEMPO were incubated with BSA or BSA+PA medium for 24 h. A , Schematic diagram showing the procedure of NRVMs. B , Intracellular total ROS using DCFH‐DA staining (upper) and mitochondrial ROS using MitoSOX staining (lower) were determined in NRVMs. C , Cell lysates of NRVMs were immunoprecipitated with TXNIP antibody, and immunoblot assays were performed using NLRP3, TXNIP, and TRX antibodies. NRVMs were treated with siTXNIP to knockdown of TXNIP. D , Triple immunofluorescence staining for TXNIP (red), NLRP3 (green), and nuclei (DAPI, blue) was performed in indicated NRVMs. E , Representative immunoblots of ASC, procapase‐1, cleaved caspase‐1 p20, IL‐1β, and IL‐18 protein in NRVMs from indicated groups. F , Oil Red O staining of NRVMs from indicated groups. Red indicates lipid droplets, blue indicates nuclei. G , qRT‐PCR detection of indicated genes related to fatty acid transport genes Cd36 and fatty acid oxidation genes Cpt1b , Acadl , and Acadvl in hearts from indicated mice (n=6 independent experiments). H , Expression level of hypertrophic marker gene BNP was determined by RT‐PCR and normalized to that of GAPDH (n=6 independent experiments). ASC indicates apoptosis‐associated speck like protein; BNP, B‐type natriuretic peptide; BSA, Bovine Serum Albumin; IL, interleukin; IP:TXNIP, immunoprecipitation:thioredoxin‐interacting protein; ns, not significant; NLRP3, nucleotide‐binding oligomerization domain‐like receptor 3; NRVM, neonatal rat ventricular myocyte; PA, palmitic acid; qRT‐PCR, quantitative real‐time polymerase chain reaction; and ROS, reactive oxygen species.

Article Snippet: Selective NLRP3 inhibitor MCC950 sodium (HY‐12815A) and mitochondria ROS scavenger MitoTEMPO (HY‐112879) were obtained from from Medchem Express (Shanghai, China).

Techniques: Incubation, Staining, Immunoprecipitation, Western Blot, Knockdown, Immunofluorescence, Quantitative RT-PCR, Expressing, Marker, Reverse Transcription Polymerase Chain Reaction, Binding Assay, Real-time Polymerase Chain Reaction

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Impeding Nucleotide‐Binding Oligomerization Domain‐Like Receptor 3 Inflammasome Ameliorates Cardiac Remodeling and Dysfunction in Obesity‐Associated Cardiomyopathy

doi: 10.1161/JAHA.124.035234

Figure Lengend Snippet: A through L , WT mice received NS or MitoTEMPO were subjected to ND or HFD feeding for 24 wks. A , Experimental schematic diagram showing the process of HFD‐induced obesity cardiomyopathy. After 12 wks of HFD feeding, MitoTEMPO was injected into obese mice at a dose of 10 mg/kg per day for 12 wks. B , C , LV fractional shortening ( B ) and ratio between mitral E wave and A wave ( C ) were measured by echocardiography in indicated mice (n=12 per group). D , Histogram of HW/TL in indicated mice. HW indicated wet weight (n=12 per group). E , Representative images of cardiac WGA staining, Oil Red O staining, and Masson trichrome staining in perivascular and interstitial area in indicated heart. F , Quantification of cardiomyocyte cross‐sectional area by WGA (n=6 per group). G , Quantification of LV collagen volume in interstitial area by Masson trichrome staining (n=6 per group). H , Quantification of myocardial lipid accumulation by Oil Red O staining (n=6 per group). I , Tissue lysate in indicated heart were immunoprecipitated with TXNIP antibody, and immunoblot assays were performed using NLRP3, TXNIP, and thioredoxin antibodies. J , K , Protein of IL‐1β ( J ) and IL‐18 ( K ) in the heart of WT mice fed an HFD or ND for 24 wks with or without MitoTEMPO treatment. IL‐1β and IL‐18 protein were detected by ELISA. Values were normalized to total protein level (n=6 per group). L , Representative immunoblots of ASC, procapase‐1, cleaved caspase‐1 p20, IL‐1β, and IL‐18 in the indicated heart. ASC indicates apoptosis‐associated speck like protein; HFD, high‐fat diet; HW/TL, heart weight/tibia length ratio; IL, interleukin; IP:TXNIP, immunoprecipitation:thioredoxin‐interacting protein; LV, left ventricular; ND, normal diet; ns, not significant; NLRP3, nucleotide‐binding oligomerization domain‐like receptor 3; NRVM, neonatal rat ventricular myocyte; ORO, Oil Red O; NS, normal saline; WGA, wheat germ agglutinin; and WT, wild type.

Article Snippet: Selective NLRP3 inhibitor MCC950 sodium (HY‐12815A) and mitochondria ROS scavenger MitoTEMPO (HY‐112879) were obtained from from Medchem Express (Shanghai, China).

Techniques: Injection, Staining, Immunoprecipitation, Western Blot, Enzyme-linked Immunosorbent Assay, Binding Assay, Saline