ROS-mediated PARP activity undermines mitochondrial function after ...
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2013 Apr 18;2(2):e000159. doi: 10.1161/JAHA.113.000159.ROS-mediated PARP activity undermines mitochondrial function after permeability transition pore opening during myocardial ischemia-reperfusion.1, , , .1Division of Neonatology, Department of Pediatrics, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA.AbstractBACKGROUND: Ischemia-reperfusion (I/R) studies have implicated oxidant stress, the mitochondrial permeability transition pore (mPTP), and poly(ADP-ribose) polymerase (PARP) as contributing factors in myocardial cell death. However, the interdependence of these factors in the intact, blood-perfused heart is not known. We therefore wanted to determine whether oxidant stress, mPTP opening, and PARP activity contribute to the same death pathway after myocardial I/R.METHODS AND RESULTS: A murine left anterior descending coronary artery (LAD) occlusion (30 minutes) and release (1 to 4 hours) model was employed. Experimental groups included controls and antioxidant-treated, mPTP-inhibited, or PARP-inhibited hearts. Antioxidant treatment prevented oxidative damage, mPTP opening, ATP depletion, and PARP activity, placing oxidant stress as the proximal death trigger. Genetic deletion of cyclophilin D (CypD(-/-)) prevented loss of total NAD(+) and PARP activity, and mPTP-mediated loss of mitochondrial function. Control hearts showed progressive mitochondrial depolarization and loss of ATP from 1.5 to 4 hours of reperfusion, but not outer mitochondrial membrane rupture. Neither genetic deletion of PARP-1 nor its pharmacological inhibition prevented the initial mPTP-mediated depolarization or loss of ATP, but PARP ablation did allow mitochondrial recovery by 4 hours of reperfusion.CONCLUSIONS: These results indicate that oxidant stress, the mPTP, and PARP activity contribute to a single death pathway after I/R in the heart. PARP activation undermines cell survival by preventing mitochondrial recovery after mPTP opening early in reperfusion. This suggests that PARP-mediated prolongation of mitochondrial depolarization contributes significantly to cell death via an energetic crisis rather than by mitochondrial outer membrane rupture.PMID:
[PubMed - indexed for MEDLINE] PMCID: PMC3647275 Identification of the area at risk (AAR) and the area of necrosis after I/R. A, AAR in wild‐ ype (WT) and experimental groups subjected to ischemia (30 minutes) followed by 4 hours of reperfusion: WT, n=8; EUK, n=6; CypD, n=6; 3AB, n=6; WT/3AB/EUK, n=6; CypD/3AB, n=7; CypD/EUK, n=8; PARP WT, n=6; PARP KO, n=6. No significant differences were detected. B, Area of necrosis in WT and experimental groups after ischemia (30 minutes) followed by 4 hours reperfusion. Representative slices from each group showing the LV not at risk (purple), the AAR (red+white), and the area of necrosis (white). *P&0.05 compared with WT. Values are means±SEMs. I/R indicates ischemia– EUK, EUK134, SODII,
CypD, cyclophilin D; 3AB, 3‐ PARP, poly(ADP‐
ibose) KO, LV, left ventricle.J Am Heart Assoc. ):e000159.Protein oxidation and DNA damage after I/R. A, Analysis of dot blots of total cell protein from the AAR after derivatization of protein carbonyls. Representative dots from a panel blot are shown: sham, n=9; WT, n=8; EUK, n=7; CypD, n=9; 3AB, n=7; PARP WT, n=7; PARP KO, n=5. B, Nuclear and mitochondrial DNA amplification in sham, WT, and experimental groups after ischemia (30 minutes) followed by reperfusion for 90 minutes: sham, n=8; WT, n=6; EUK, n=5; CypD, n=5; 3AB, n=5; PARP WT, n=7; PARP KO, n=7. Values expressed relative to sham. Representative ethidium bromide–stained gels of the long and short mitochondrial products are shown. *P&0.05 compared with sham, # different compared with WT. Values are means±SEMs. I/R indicates ischemia– AAR, WT, EUK, EUK134, SODII,
CypD, cyclophilin D; 3AB, 3‐ PARP, poly(ADP‐
ibose) KO, knockout.J Am Heart Assoc. ):e000159.Poly(ADP‐
ibosylation) (PAR) after I/R. Immunoblotting of total cell protein from AAR of hearts. A, Wild‐ ype (WT) hearts subjected to sham occlusion, ischemia (30 minutes) without reperfusion, or ischemia (30 minutes) followed by 15, 60, or 90 minutes of reperfusion, n=5 all groups. B, WT, EUK, CypD‐KO, 3AB, and PARP‐KO hearts after ischemia (30 minutes) and 90 minutes of reperfusion: sham, n=12; WT, n=11; EUK, n=9; CypD, n=10; 3AB, n=10; PARP WT, n=6; PARP KO, n=6. Gel bands were analyzed by densitometry and normalized to tubulin as a loading control. Total PAR protein levels are expressed relative to sham levels. *P&0.05 compared with sham and # compared with WT controls. Values are means±SEMs. I/R indicates ischemia– AAR, EUK, EUK134, SODII,
CypD, cyclophilin D; 3AB, 3‐ PARP, poly(ADP‐
ibose) KO, knockout.J Am Heart Assoc. ):e000159.NAD(H) depletion after I/R. NAD(H) levels in the AAR. A, Wild‐ ype (WT) hearts subjected to sham occlusion, ischemia (30 minutes) without reperfusion, or ischemia (30 minutes) followed by 15, 60, or 90 minutes of reperfusion, n=5 all groups. B, WT, EUK, CypD‐KO, 3AB, and PARP‐KO hearts after 30 minutes of ischemia and 90 minutes of reperfusion: sham, n=6; WT, n=9; EUK, n=7; CypD, n=7; 3AB, n=8; PARP WT, n=6; PARP KO, n=6. NADH levels were normalized to total protein concentration and then normalized to sham levels. *P&0.05 compared with sham and # compared with WT controls. Values are means±SEMs. I/R indicates ischemia– AAR, EUK, EUK134, SODII,
CypD, cyclophilin D; 3AB, 3‐ PARP, poly(ADP‐
ibose) KO, knockout.J Am Heart Assoc. ):e000159.Mitochondrial NAD(H) after I/R. A, NAD(H) in mitochondrial isolates following ischemia (30 minutes) followed by 60 minutes of reperfusion: sham, n=7; WT, n=6; EUK, n=6; CypD, n=7; 3AB, n=8; PARP WT, n=10; PARP KO, n=10. Values are expressed relative to sham group. B, NAD(H) in mitochondrial fractions assayed after 1 and 4 hours of reperfusion, n=6 all groups. Values are expressed relative to sham group. *P&0.05 compared with sham and # compared with WT controls. Values are means±SEMs. I/R indicates ischemia– AAR, EUK, EUK134, SODII,
CypD, cyclophilin D; 3AB, 3‐ PARP, poly(ADP‐
ibose) KO, knockout.J Am Heart Assoc. ):e000159.ATP and total adenine nucleotide levels after I/R. Total ATP levels in the AAR of hearts following ischemia (30 minutes) followed by (A) 1.5 hours or (B) 4 hours of reperfusion. C, Total adenine nucleotide levels in the AAR of hearts following ischemia (30 minutes) followed by 4 hours of reperfusion. At 1.5 hours, sham, n=6; WT, n=5; EUK, n=6; CypD, n=6; 3AB, n=6; PARP WT, n=6; PARP KO, n=6. At 4 hours, sham, n=5; WT, n=7; EUK, n=6; CypD, n=6; 3AB, n=6; PARP WT, n=6; PARP KO, n=6. Total adenine nucleotide levels, sham, n=8; WT, n=8; 3AB, n=8. Values are expressed relative to sham group. *P&0.05 compared with sham and # compared with WT controls. Values are means±SEMs. I/R indicates ischemia– AAR, WT, EUK, EUK134, SODII,
CypD, cyclophilin D; 3AB, 3‐ PARP, poly(ADP‐
ibose) KO, knockout.J Am Heart Assoc. ):e000159.TMRE localization, stability, and responses to CCCP ex vivo. A, Mitochondrial‐ argeted GFP‐expressing hearts were loaded with TMRE in vivo, excised, and sliced. Colocalization of GFP and TMRE fluorescence was confirmed by 2‐photon microscopy. B, Hearts were loaded with TMRE, excised, sliced, and imaged during CCCP (50 or 250 μmol/L) treatment for 15 minutes. C, Hearts were loaded with TMRE, excised, sliced, and imaged while being maintained in ice‐cold PBS for 30 minutes followed by 15 minutes of treatment with 250 μmol/L CCCP, n=5 all groups. TMRE indicates tetramethyl CCCP, carbonyl cyanide m‐ch GFP, green PBS, phosphate‐uffered saline.J Am Heart Assoc. ):e000159.Mitochondrial polarization in relation to plasma membrane rupture after I/R. TMRE fluorescence and calcein‐AM staining at different times after reperfusion. TMRE was loaded into beating hearts for 15 minutes. TMRE and calcein fluorescence change in hearts subjected to ischemia (30 minutes) followed by reperfusion for 15 minutes or 4 hours, n=5 all groups. *P&0.05 compared with sham and # compared with 15‐minute time. Values are means±SEMs. I/R indicates ischemia– TMRE, tetramethyl AAR, LV, left ventricle.J Am Heart Assoc. ):e000159.Mitochondrial polarization after I/R. Hearts were loaded with TMRE at different points after reperfusion. Hearts were then retroperfused with Hoechst to delineate the AAR, sliced, and imaged. Total fluorescence of the AAR was compared with the area not at risk and normalized to sham hearts. Fluorescence changes from genetically matched WT, 3AB, PARP‐1‐KO, and CypD‐KO hearts were compared after (A) 1.5 hours or (B) 4 hours of reperfusion. Included are representative heart slices showing TMRE voids in the AAR, n=5 all groups. *P&0.05 compared with sham and # compared with WT controls. Values are means±SEMs. I/R indicates ischemia– TMRE, tetramethyl AAR, WT, CypD, cyclophilin D; 3AB, 3‐ PARP, poly(ADP‐
ibose) KO, knockout.J Am Heart Assoc. ):e000159.Cardiac troponin release in relation to cytochrome c or AIF translocation after I/R. A, Cardiac troponin in circulating plasma as a measure of plasma membrane rupture after 1.5 and 4 hours of reperfusion and in EUK, CypD‐KO, 3AB, and PARP‐KO hearts at 4 hours of reperfusion. B, Mitochondrial AIF, mitochondrial cytochrome c, and cytosolic AIF and cytochrome c in cellular fractions in sham hearts or after ischemia (30 minutes) followed by reperfusion for 1 hour (IR1h), 4 hours (IR4h), 16 hours (IR16h), or ex vivo hearts allowed to autolyse for 1 hour (AL): sham, n=6; IR1.5h, n=5; IR4h, n=6; IR16h, n=6; AL, n=5. Mitochondrial values are normalized to cytochrome oxidase subunit 4 (Cox IV), and cytosolic values are normalized to GAPDH. *P&0.05 compared with sham. Values are means±SEMs. AIF indicates apoptosis‐ I/R, ischemia– AAR, WT, EUK, EUK134, SODII,
CypD, cyclophilin D; 3AB, 3‐ PARP, poly(ADP‐
ibose) KO, Cox IV, cytochrome c oxidase subunit 4.J Am Heart Assoc. ):e000159.Model of myocardial I/R‐induced cell death. I/R indicates ischemia– ROS, rea mPTP, mitochondrial permeabi PARP, poly(ADP‐
ibose) polymerase.J Am Heart Assoc. ):e000159.Publication TypesMeSH TermsSubstancesGrant SupportFull Text SourcesOther Literature Sources
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Genetic deletion of the mitochondrial phosphate carrier desensitizes the mitochondrial permeability transition pore and causes cardiomyopathy.
The mitochondrial phosphate carrier (PiC) is critical for ATP synthesis by serving as the primary means for mitochondrial phosphate import across the inner membrane. In addition to its role in energy production, PiC is hypothesized to have a role in cell death as either a component or a regulator of the mitochondrial permeability transition pore (MPTP) complex. Here, we have generated a mouse model with inducible and cardiac-specific deletion of the Slc25a3 gene (PiC protein). Loss of PiC protein did not prevent MPTP opening, suggesting it is not a direct pore-forming component of this complex. However, Slc25a3 deletion in the heart blunted MPTP opening in response to Ca(2+) challenge and led to a greater Ca(2+) uptake capacity. This desensitization of MPTP opening due to loss or reduction in PiC protein attenuated cardiac ischemic-reperfusion injury, as well as partially protected cells in culture from Ca(2+) overload induced death. Intriguingly, deletion of the Slc25a3 gene from the heart long-term resulted in profound hypertrophy with ventricular dilation and depressed cardiac function, all features that reflect the cardiomyopathy observed in humans with mutations in SLC25A3. Together, these results demonstrate that although the PiC is not a direct component of the MPTP, it can regulate its activity, suggesting a novel therapeutic target for reducing necrotic cell death. In addition, mice lacking Slc25a3 in the heart serve as a novel model of metabolic, mitochondrial-driven cardiomyopathy.
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丁香园旗下网站Calcium-induced cardiac mitochondrial dysfunction is predominantly ...
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):333-8. doi: 10.1016/j.arcmed.. Epub
2012 Jul 21.Calcium-induced cardiac mitochondrial dysfunction is predominantly mediated by cyclosporine A-dependent mitochondrial permeability transition pore.1, , , , .1Cardiac Electrophysiology Research and Training Center, Faculty of Medicine, Chiang Mai University, Thailand.AbstractBACKGROUND AND AIMS: Cardiac mitochondrial Ca(2+) overload plays a critical role in mechanical and electrical dysfunction leading to cardiac cell death and fatal arrhythmia. Because Ca(2+) overload is related to mitochondrial permeability transition, reactive oxygen species (ROS) production and membrane potential (ΔΨm) dissipation, we probed the mechanistic association between Ca(2+) overload, oxidative stress, mitochondrial permeability transition pore (mPTP) and mitochondrial calcium uniporter (MCU) in isolated cardiac mitochondria.METHODS: Various concentrations of Ca(2+) (5-200 μM) were used to induce mitochondrial dysfunction. Cyclosporin A (CsA, an mPTP blocker) and Ru360 (an MCU blocker) were used to test its protective effects on Ca(2+)-induced mitochondrial dysfunction.RESULTS: High concentrations of Ca(2+) (≥100 μM) caused overt mitochondrial swelling and ΔΨm collapse. However, only slight increases in ROS production were detected. Blocking the MCU by Ru360 is less effective in protecting mitochondrial dysfunction.CONCLUSIONS: A dominant cause of Ca(2+)-induced cardiac mitochondrial dysfunction was mediated through the mPTP rather than MCU. Therefore, CsA could be more effective than Ru360 in preventing Ca(2+)-induced cardiac mitochondrial dysfunction.Copyright (C) 2012 IMSS. Published by Elsevier Inc. All rights reserved.PMID:
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External link. Please review our .Loss of DJ-1 does not affect mitochondrial respiration but increase...
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):e40501. doi: 10.1371/journal.pone.0040501. Epub
2012 Jul 9.Loss of DJ-1 does not affect mitochondrial respiration but increases ROS production and mitochondrial permeability transition pore opening.1, , , , .1Center for Neurologic Diseases, Brigham and Women's Hospital, Program in Neuroscience, Harvard Medical School, Boston, Massachusetts, United States of America.AbstractBACKGROUND: Loss of function mutations in the DJ-1 gene have been linked to recessively inherited forms of Parkinsonism. Mitochondrial dysfunction and increased oxidative stress are thought to be key events in the pathogenesis of Parkinson's disease. Although it has been reported that DJ-1 serves as scavenger for reactive oxidative species (ROS) by oxidation on its cysteine residues, how loss of DJ-1 affects mitochondrial function is less clear.METHODOLOGY/PRINCIPAL FINDINGS: Using primary mouse embryonic fibroblasts (MEFs) or brains from DJ-1-/- mice, we found that loss of DJ-1 does not affect mitochondrial respiration. Specifically, endogenous respiratory activity as well as basal and maximal respiration are normal in intact DJ-1-/- MEFs, and substrate-specific state 3 and state 4 mitochondrial respiration are also unaffected in permeabilized DJ-1-/- MEFs and in isolated mitochondria from the cerebral cortex of DJ-1-/- mice at 3 months or 2 years of age. Expression levels and activities of all individual complexes composing the electron transport system are unchanged, but ATP production is reduced in DJ-1-/- MEFs. Mitochondrial transmembrane potential is decreased in the absence of DJ-1. Furthermore, mitochondrial permeability transition pore opening is increased, whereas mitochondrial calcium levels are unchanged in DJ-1-/- cells. Consistent with earlier reports, production of reactive oxygen species (ROS) is increased, though levels of antioxidative enzymes are unaltered. Interestingly, the decreased mitochondrial transmembrane potential and the increased mitochondrial permeability transition pore opening in DJ-1-/- MEFs can be restored by antioxidant treatment, whereas oxidative stress inducers have the opposite effects on mitochondrial transmembrane potential and mitochondrial permeability transition pore opening.CONCLUSIONS/SIGNIFICANCE: Our study shows that loss of DJ-1 does not affect mitochondrial respiration or mitochondrial calcium levels but increases ROS production, leading to elevated mitochondrial permeability transition pore opening and reduced mitochondrial transmembrane potential.PMID:
[PubMed - indexed for MEDLINE] PMCID: PMC3392228 (A) Endogenous respiratory activity in DJ-1-/- and +/+ MEFs. Representative oxygraphs of DJ-1-/- and +/+ MEFs energized with glucose (10 mM) are shown on the left. The bar graph on the right shows oxygen consumption, which represents the endogenous respiratory activity in DJ-1-/- and +/+ MEFs. The data were obtained from three independent experiments using primary MEFs obtained from 3 individual embryos per genotype. (B) Oxygen consumption rate (OCR) profile in DJ-1-/- and +/+ MEFs. OCR profile expressed as pMolesO2/min in control and DJ-1-/- cells are shown on the left. Arrows indicate the time of addition of oligomycin (Oligo, 1 uM), FCCP (4 uM) and rotenone (100 nM). The bar graph on the right shows OCRs normalized to protein concentration after subtraction of rotenone insensitive OCR (nonmitochondrial respiration), under basal condition, after addition of oligomycin (Oligo, 1 uM, proton leak) or FCCP (4 uM, maximal respiration). The data were obtained from three independent experiments using primary MEFs obtained from 6 individual embryos. (C) Energized respiration in DJ-1-/- and +/+ MEFs. Representative traces of respiration rates in the mitochondria in DJ-1-/- and +/+ MEFs are shown on the left. Arrows indicate the application of substrates (complex I: 10 mM glutamate/malate (GM), complex II: succinate (Succ, 10 mM), complex III/IV: 1 mM TMPD/1 mM ascorbate (TMPD)) in the presence of ADP (1 mM) and oligomycin (oligo). The bar graphs on the right show state 3 respiratory activity for complex I, II and III/IV in DJ-1-/- and +/+ MEFs permeabilized with digitonin. The number shown in the panel indicates the number of embryos used to derive primary MEFs per genotype, and the data were obtained from three independent experiments. All data are expressed as the mean ± S.E.PLoS One. ):e40501.(A) Energized respiration in mitochondria isolated from the cerebral cortex of DJ-1-/- and +/+ mice at 3 months of age. Representative traces of respiration rates in mitochondria isolated from the cortex of DJ-1-/- and littermate control mice are shown on the left. Arrows indicate the time of the application of substrates (complex I: 10 mM glutamate/malate (GM), complex II: succinate (Succ, 10 mM), complex III/IV: 1 mM TMPD/1 mM ascorbate (TMPD)) in the presence of ADP (1 mM). The bar graphs on the right show state 3 and state 4 respiratory activities for complex I, II and III/IV in isolated mitochondria from the cortex of DJ-1-/- and +/+ mice at the age of 3 months. (B) Energized respiration in mitochondria isolated from the cortex of 24–26 months old DJ-1-/- and +/+ mice. Representative traces of respiration rates in mitochondria isolated from the cortex of 24–26 months old DJ-1-/- and control mice are shown on the left. Arrows indicate the time of the application of substrates (complex I: 10 mM glutamate/malate (GM), complex II: succinate (Succ, 10 mM), complex III/IV: 1 mM TMPD/1 mM ascorbate (TMPD)) in the presence of ADP (1 mM). The bar graphs on the right show state 3 and state 4 respiratory activities for complex I, II and III/IV in isolated mitochondria from the cortex of DJ-1-/- and control mice at 24–26 months of age. The number shown in the panel indicates the number of mice used in the study. All data are expressed as the mean ± S.E.PLoS One. ):e40501.(A, B) Western analysis of each subunit in the oxidative phosphorylation (OXPHOS) complex in DJ-1-/- and +/+ MEFs. (A) Representative western blot showing relative expression of each subunit. Tubulin was used as loading control. Non-specific bands are marked by asterisk. (B) The bar graph shows the quantification and normalization of the expression level of each subunit using tubulin as loading control. (C) Enzymatic activities of complexes I, II and IV of the mitochondrial electron transport system, as measured by spectrophotometric assays and after normalization to citrate synthase activity (CS). (D) The bar graph shows decreased ATP concentrations in DJ-1-/- MEFs compared to control cells. The number shown in the panel indicates the number of embryos used to derive primary MEFs per genotype, and the data were obtained from three independent experiments. All data are expressed as mean ± SEM. *p&0.05.PLoS One. ):e40501.(A, B) Confocal microscopic analysis. (A) Representative confocal microscopic images of DJ-1-/- and +/+ MEFs after staining with TMRM (50 nM, red) and Mitotracker Green (200 nM) in the presence or absence of oligomycin (Olig, 1 uM) or FCCP (10 uM). The intensity of TMRM reflects the level of ΔΨm, whereas the intensity of Mitotracker Green is not affected by transmembrane potential. Insets in panels indicate higher power views of the boxed area. Scale bar: 10 um. (B) The bar graph shows quantification of TMRM signal in DJ-1-/- and +/+ MEFs in the presence or absence of oligomycin or FCCP. The TMRM signal is reduced in DJ-1-/- cells relative to wild-type cells, whereas the TMRM signal is increased or decreased in both DJ-1-/- and +/+ cells following oligomycin or FCCP treatment, respectively. The number shown in the panel indicates the number of cells quantified per genotype in the study. (C, D) FACS analysis. (C) Representative flow cytometric dot plots show the intensity of TMRM signal in DJ-1-/- and +/+ MEFs following incubation with TMRM (50 nM) in the presence or absence of oligomycin (1 uM) or FCCP (10 uM). (D) The bar graph shows quantification of TMRM signal measured by FACS analysis in DJ-1-/- and +/+ MEFs. The number shown in the panel indicates the number of embryos used to derive primary MEFs per genotype, and the data were obtained from five independent experiments. All data are expressed as mean ± SEM. **p&0.01, ***p&0.001.PLoS One. ):e40501.(A, B) Confocal microscopy analysis. (A) Representative confocal microscopic images of DJ-1-/- and +/+ MEFs after incubation with calcein-AM (1 uM, green) and Mitotracker Red (150 nM) in the presence or absence of Co2+ (1 mM), which quenches calcein fluorescence (green) outside of mitochondria. Mitotracker Red confirms the localization of calcein fluorescence in mitochondria. Insets indicate higher power views of the boxed area in the panel. The calcein fluorescence in mitochondria is lower in DJ-1-/- cells in the presence of Co2+. In the absence of Co2+, calcein fluorescent signals are very intense and are present in the entire cell, and there are no genotypic differences. Scale bar: 10 um. (B) The bar graph shows quantification of calcein fluorescence in DJ-1-/- and +/+ cells in the presence or absence of Co2+. The number shown in the panel indicates the number of cells quantified per genotype in the study. (C, D) FACS analysis. (C) Representative flow cytometric dot plots show the intensity of calcein signal in DJ-1-/- and +/+ MEFs following incubation with calcein-AM (1 uM) in the presence or absence of Co2+ (1 mM). (D) The bar graph of calcein signal measured by FACS analysis shows reduced calcein signal in DJ-1-/- MEFs in the presence of Co2+. The number shown in the panel indicates the number of embryos used to derive primary MEFs per genotype, and the data were obtained from five independent experiments. All data are expressed as mean ± SEM. *p&0.05, ***p&0.001.PLoS One. ):e40501.(A) Representative Fura-2 images of Ca2+ responses following FCCP treatment in DJ-1-/- and +/+ MEFs. Fura-2 ratios at 340/387 are shown at time points indicated. The green fluorescence images show the shape of the DJ-1-/- and +/+ MEFs. The pseudocolor calibration scale for 340/387 ratios is shown on the right. FCCP (1 uM) was added at t = 25 s. (B) Time course of cytosolic [Ca2+] rise following FCCP treatment in DJ-1-/- and +/+ MEFs. (C) The basal and the peak value of cytosolic calcium rise following FCCP are the same in DJ-1-/- and +/+ MEFs. The number shown in the panel indicates the number of embryos used to derive primary MEFs, and the data were obtained from three independent experiments. All data are expressed as mean ± SEM.PLoS One. ):e40501.(A, B) Confocal microscopy analysis of ROS concentration. (A) Representative confocal live cell images of DJ-1-/- and +/+ MEFs after incubation with Mitotracker Green (200 nM) and Amplex Red (2.5 uM), DHEt (2.5 uM) or MitoSOX Red (2.5 uM). Scale bar: 10 um. (B) The bar graph shows the quantification and the increase of Amplex Red, DHEt or MitoSOX Red fluorescence in DJ-1-/- cells compared to control cells. The number shown in the panel indicates the number of cells quantified per genotype in the study. (C) Kinetics analysis of ROS production. The time course of the fluorescence changes in DJ-1-/- and +/+ MEFs labeled with Amplex Red (upper), DHEt (middle), or Mitotracker CM-H2XROS (lower) is shown. The bar graph at the bottom shows quantitative analysis of fluorescence changes, indicating significant increases of fluorescence signals of Amplex Red, DHEt and Mitotracker CM-H2XROS in DJ-1-/- MEFs. The number shown in the panel indicates the number of embryos used to derive primary MEFs per genotype, and the data were obtained from four independent experiments. (D) Kinetics of H2O2 production in isolated mitochondria measured by following Amplex Red fluorescence over time showing an increase of its production in DJ-1-/- MEFs. The number shown in the panel indicates the number of embryos used to derive primary MEFs per genotype, and the data were obtained from three independent experiments. All data are expressed as the mean ± S.E. *p&0.05, ***p&0.001.PLoS One. ):e40501.(A) Representative western blot showing expression levels of Catalase, G6PDH, SOD1 and SOD2. Tubulin was used as loading control. (B) The bar graph shows the quantification of the level of each protein normalized to tubulin. The number shown in the panel indicates the number of embryos used to derive primary MEFs per genotype, and the data were obtained from three independent experiments. All data are expressed as mean ± SEM.PLoS One. ):e40501.(A, B) Confocal microscopic analysis. (A) Representative confocal live cell images of DJ-1-/- and +/+ MEFs stained with TMRM (50 nM, red) and Mitotracker Green (200 nM) after incubation with or without glutathione (Glu, 10 mM, 24 hr) or NAC (20 mM, 24 hr). Insets show higher power views of the boxed area in the panel. Scale bar: 10 um. (B) The bar graph shows quantification of TMRM signal in DJ-1-/- and +/+ MEFs after incubation with or without glutathione (Glu, 10 mM, 24 hr) or NAC (20 mM, 24 hr). The TMRM signal is reduced in DJ-1-/- cells under basal conditions, whereas the TMRM signal is increased in DJ-1-/- after incubation with antioxidants. The number shown in the panel indicates the number of cells quantified per genotype in the graph. (C, D) FACS analysis. (C) Representative flow cytometric dot plots show the intensity of TMRM signal in DJ-1-/- and +/+ MEFs following incubation with TMRM (50 nM) after incubation with or without glutathione (Glu, 10 mM, 24 hr) or NAC (20 mM, 24 hr). (D) The bar graph shows quantification of TMRM signal in DJ-1-/- and +/+ MEFs and the rescue of the decrease of the TMRM fluorescence in DJ-1-/- cells after incubation with antioxidant molecules. The number shown in the panel indicates the number of embryos used to derive primary MEFs per genotype, and the data were obtained from five independent experiments. All data are expressed as mean ± SEM. *p&0.05, **p&0.01, ***p&0.001.PLoS One. ):e40501.(A, B) Confocal microscopic analysis. (A) Representative confocal live cell images of DJ-1-/- and +/+ MEFs stained with TMRM (50 nM, red) and Mitotracker Green (200 nM) after incubation with or without H2O2 (500 uM, 3 hr) or pyocyanin (100 uM, 24 hr). Insets show higher power views of the boxed area in the panel. Scale bar: 10 um. (B) The bar graph shows quantification of TMRM signal in DJ-1-/- and +/+ MEFs after incubation with or without H2O2 (500 uM, 3 hr) or pyocyanin (100 uM, 24 hr). The TMRM signal is markedly reduced in DJ-1+/+ cells after induction of oxidative stress. The number shown in the panel indicates the number of cells quantified per genotype in the graph. (C, D) FACS analysis. (C) Representative flow cytometry dot plots show the intensity of TMRM signal in DJ-1-/- and +/+ MEFs following incubation with TMRM (50 nM) after incubation with or without H2O2 (500 uM, 3 hr) or pyocyanin (100 uM, 24 hr). (D) The bar graph shows quantification of TMRM signal in DJ-1-/- and +/+ MEFs. The TMRM fluorescence in DJ-1+/+ cells is decreased after incubation with oxidative stress inducers. The number shown in the panel indicates the number of embryos used to derive primary MEFs per genotype, and the data were obtained from three independent experiments. All data are expressed as mean ± SEM. *p&0.05, **p&0.01, ***p&0.001.PLoS One. ):e40501.(A, B) Confocal microscopic analysis. (A) Representative confocal live cell images of DJ-1-/- and +/+ MEFs stained with calcein-AM (1 uM, green) and Mitotracker Red (150 nM) in the presence of Co2+ (1 mM) after incubation with or without glutathione (Glu, 10 mM, 24 hr) or NAC (20 mM, 24 hr). Insets show higher power views of the boxed area in the panel. Scale bar: 10 um. (B) The bar graph shows quantification of calcein signal in DJ-1-/- and +/+ MEFs after incubation with or without glutathione (Glu, 10 mM, 24 hr) or NAC (20 mM, 24 hr). The calcein signal is reduced in DJ-1-/- cells under basal conditions, whereas this signal is increased in DJ-1-/- after incubation with antioxidants. The number shown in the panel indicates the number of cells quantified per genotype in the study. (C, D) FACS analysis. (C) Representative flow cytometric dot plots show the intensity of calcein signal in DJ-1-/- and +/+ MEFs following incubation with calcein-AM (1 uM) in the presence of Co2+ (1 mM) after incubation with or without glutathione (Glu, 10 mM, 24 hr) or NAC (20 mM, 24 hr). (D) The bar graph shows quantification of calcein signal in DJ-1-/- and +/+ MEFs and the reversal of the decrease of calcein fluorescence in DJ-1-/- cells after incubation with antioxidant molecules. The number shown in the panel indicates the number of embryos used to derive primary MEFs per genotype, and the data were obtained from three independent experiments. All data are expressed as mean ± SEM. *p&0.05, **p&0.01.PLoS One. ):e40501.DJ-1+/+ MEFs. (A, B) Confocal microscopic analysis. (A) Representative confocal live cell images of DJ-1-/- and +/+ MEFs stained with calcein-AM (1 uM, green) and Mitotracker Red (150 nM) in the presence of Co2+ (1 mM) after incubation with or without H2O2 (500 uM, 3 hr) or pyocyanin (100 uM, 24 hr). Insets show higher power views of the boxed area in the panel. Scale bar: 10 um. (B) The bar graph shows quantification of calcein signal in DJ-1-/- and +/+ MEFs after incubation with or without H2O2 (500 uM, 3 hr) or pyocyanin (100 uM, 24 hr). Following treatment, the calcein signal is markedly reduced in DJ-1+/+ cells, and is also reduced in DJ-1-/- cells. The number shown in the panel indicates the number of cells quantified per genotype in the graph. (C, D) FACS analysis. (C) Representative flow cytometry dot plots show the intensity of calcein signal in DJ-1-/- and +/+ MEFs following incubation with calcein-AM (1 uM, green) in the presence of Co2+ (1 mM) after incubation with or without H2O2 (500 uM, 3 hr) or pyocyanin (100 uM, 24 hr). (D) The bar graph shows quantification of calcein signal in DJ-1-/- and +/+ MEFs. The calcein fluorescence in DJ-1+/+ cells is decreased after incubation with oxidative stress inducers. The number shown in the panel indicates the number of embryos used to derive primary MEFs per genotype, and the data were obtained from three independent experiments. All data are expressed as mean ± SEM. *p&0.05, **p&0.01, ***p&0.001.PLoS One. ):e40501.Publication TypesMeSH TermsSubstancesGrant SupportFull Text SourcesOther Literature SourcesMolecular Biology DatabasesMiscellaneous
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