Journal: Advanced Science

Article Title: SIRT3‐Mediated Deacetylation of DRP1 K711 Prevents Mitochondrial Dysfunction in Parkinson's Disease

doi: 10.1002/advs.202411235

Figure Lengend Snippet: SIRT‐3 regulates mitochondrial function and morphology via K711 acetylation of DRP1. A) Co‐immunoprecipitation assay showing the interaction between DRP1 and SIRT3 in HeLa cells. B) Bimolecular fluorescence complementation (BiFC) assay confirmed the interaction between DRP1 and SIRT3 in HeLa cells. Fusion constructs of DRP1‐HA and SIRT3‐FLAG were co‐expressed, and green fluorescence indicates protein interaction. Scale bar: 20 µm. C) The schematic diagram of the FLIM – FRET experiment principle for CFP – DRP1 and YFP – SIRT3. Representative images showing the lifetime distribution. D)Time‐correlated single‐photon counting‐fluorescence lifetime imaging microscopy (TCSPC FLIM) with Förster resonance energy transfer (FRET) further confirmed the interaction between DRP1 and SIRT3. Comparison of the decay data and fitting curves of cells expressing CFP alone and those co‐expressing CFP and EYFP confirmed FRET, indicating the interaction between DRP1 and SIRT3. Scale bar: 0.03 mm. E) Co‐immunoprecipitation experiments using shSIRT3 or SIRT3‐overexpressing cell lines co‐transfected with DRP1‐Flag revealed the regulatory role of SIRT3 in DRP1 acetylation. F–H) Western blotting analysis showing the impact of SIRT3 overexpression or knockdown on the acetylation levels of DRP1 at K711 in HeLa cells under the condition of H₂O₂‐induced oxidative stress. Data are represented as the mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 versus indicated group; ns , not significant. I) After being co‐transfected with the SIRT3 and K711Q plasmids, the CCK‐8 assay was conducted to show the protective effects of SIRT3 against H₂O₂‐induced cell death in HeLa cells. Data are represented as the mean ± SD (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 versus indicated group; ns , not significant. J–M) After being co‐transfected with the SIRT3 and K711Q plasmids and subsequently treated with H₂O₂, Western blotting analysis was performed to detect the expression levels of cleaved caspase‐3, Bax, and Bcl‐2 in HeLa cells, which confirmed the protective effects of SIRT3 against H₂O₂‐induced apoptosis in HeLa cells. Data are represented as the mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 versus indicated group; ns , not significant. N,O) Immunofluorescence staining with Tom20 antibody revealed the impact of SIRT3 on mitochondrial morphology in HeLa cells. Quantification of mitochondrial AR and FF is shown. Scale bar: 10 µm. Data are represented as the mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 versus indicated group; ns , not significant. Statistical analysis results are presented in Table (Supporting Information).

Article Snippet: The recombinant Sirt3 protein, sourced from MedChemExpress (HY‐P701609), was tag‐free.

Techniques: Co-Immunoprecipitation Assay, Bimolecular Fluorescence Complementation Assay, Construct, Fluorescence, Imaging, Microscopy, Förster Resonance Energy Transfer, Comparison, Expressing, Immunoprecipitation, Transfection, Western Blot, Over Expression, Knockdown, CCK-8 Assay, Immunofluorescence, Staining

Journal: Advanced Science

Article Title: SIRT3‐Mediated Deacetylation of DRP1 K711 Prevents Mitochondrial Dysfunction in Parkinson's Disease

doi: 10.1002/advs.202411235

Figure Lengend Snippet: SIRT3 protects against MPP + ‐induced apoptosis by regulating DRP1 K711 acetylation. A–D) Western blotting analysis of the effects of SIRT3 overexpression on acetylated lysine, DRP1 K711 , and SIRT3 levels in SH‐SY5Y cells after MPP + treatment. Data are represented as the mean ± SD (n = 3). *P < 0.05, **P < 0.01, and ***P < 0.001 versus indicated group; ns , not significant. E–G) Mitochondrial morphology in SH‐SY5Y cells after MPP + treatment was assessed via MitoTracker staining. Quantification of mitochondrial AR and FF is shown. Scale bars: 10 µm; 5 µm (zoomed). Data are represented as the mean ± SD (n = 3). *P < 0.05 and **P < 0.01 versus indicated group; ns , not significant. H,I) Mitochondrial membrane potential (ΔΨm) in SH‐SY5Y cells after MPP+ treatment evaluated via TMRE staining. Scale bar: 10 µm. Data are represented as the mean ± SD (n = 3). **P < 0.01 and ***P < 0.001 versus indicated group. J,K) ROS generation in MPP+ treated SH‐SY5Y cells assessed via CellROX Deep Red staining. Scale bar: 80 µm. Data are represented as the mean ± SD (n = 3). ***P < 0.001 and ****P < 0.0001 versus indicated group. L) TUNEL analysis of MPP+ treated SH‐SY5Y cell apoptosis. Scale bar: 160 µm. M) Quantification of TUNEL‐positive cells. Data are represented as the mean ± SD (n = 3). ***P < 0.001 versus indicated group. Statistical analysis results are presented in Table (Supporting Information).

Article Snippet: The recombinant Sirt3 protein, sourced from MedChemExpress (HY‐P701609), was tag‐free.

Techniques: Western Blot, Over Expression, Staining, Membrane, TUNEL Assay

Journal: Advanced Science

Article Title: SIRT3‐Mediated Deacetylation of DRP1 K711 Prevents Mitochondrial Dysfunction in Parkinson's Disease

doi: 10.1002/advs.202411235

Figure Lengend Snippet: SIRT3 agonist HKL protects dopaminergic neurons and alleviates motor dysfunction in the MPTP‐induced PD mouse model by modulating DRP1 acetylation. A) Experimental timeline for the administration of HKL and MPTP to establish a PD mouse model and subsequent behavioral testing. B–F) Western blotting analysis of DRP1 K711 , DRP1, TH, and SIRT3 levels in SNc brain tissue samples of MPTP‐induced PD model mice treated with the SIRT3 agonist, HKL. Data are represented as the mean ± SD (n = 6). *P < 0.05, **P < 0.01, and ****P < 0.0001 versus indicated group. G–I) Immunofluorescence staining and analysis of TH and SIRT3 expression levels in the SNc region of HKL‐treated MPTP‐induced C57BL/6J PD model mice. Data are represented as the mean ± SD (n = 3). ****P < 0.0001 versus indicated group. J) Rotarod test analysis of motor function in MPTP‐induced PD mice treated with HKL. Data are represented as the mean ± SD (n = 8). *P < 0.05 and ***P < 0.001 versus indicated group. K) Test diagram from the Catwalk system. The system shows green paw prints and records parameters after recognition. L–N) Catwalk gait analysis of cadence, duration, and average speed in HKL‐treated MPTP‐induced PD mice. Data are represented as the mean ± SD (n = 8). *P < 0.05 and **P < 0.01 versus indicated group.

Article Snippet: The recombinant Sirt3 protein, sourced from MedChemExpress (HY‐P701609), was tag‐free.

Techniques: Western Blot, Immunofluorescence, Staining, Expressing

Journal: Advanced Science

Article Title: SIRT3‐Mediated Deacetylation of DRP1 K711 Prevents Mitochondrial Dysfunction in Parkinson's Disease

doi: 10.1002/advs.202411235

Figure Lengend Snippet: SIRT3 knockout exacerbates dopaminergic neuron loss and motor deficits in a PD mouse model. A) Generation of TH‐specific SIRT3 ‐knocked‐out mice via stereotaxic injection of pAAV‐TH‐Cre‐WPRE‐hGHpA virus into the SNc of SIRT3 flox/flox mice. MPTP was administered to establish a PD mouse model, which was subjected to behavioral testing. B–D) Western blotting analysis of DRP1 and total acetylation levels in the SNc brain tissues of MPTP‐induced SIRT3 ‐knocked‐out PD mice. Data are represented as the mean ± SD (n = 6). **P < 0.01, ***P < 0.001 and ****P < 0.0001 versus indicated group. E–G) Western blotting analysis of SIRT3 and DRP1 K711 levels in the SNc brain tissues of MPTP‐induced PD mice with SIRT3 CKO. Data are represented as the mean ± SD (n = 6). ***P < 0.001 and ****P < 0.0001 versus indicated group. H,I) Immunofluorescence analysis of TH and SIRT3 expression levels in the SNc region of MPTP‐induced PD mice with SIRT3 CKO. Data are represented as the mean ± SD (n = 3). *P < 0.05, **P < 0.01, and ***P < 0.001 versus indicated group. J) Rotarod test analysis of motor function in MPTP‐induced PD mice with SIRT3 CKO. Data are represented as the mean ± SD (n = 5). *P < 0.05, **P < 0.01, and ****P < 0.0001 versus indicated group. K) Test diagram from the Catwalk system. The system shows green paw prints and records parameters after recognition. L–N) Catwalk gait analysis of cadence, duration, and average speed in MPTP‐induced PD mice with SIRT3 CKO. Data are represented as the mean ± SD (n = 5). *P < 0.05, ***P < 0.001, and ****P < 0.0001 versus indicated group. Statistical analysis results are presented in Table (Supporting Information).

Article Snippet: The recombinant Sirt3 protein, sourced from MedChemExpress (HY‐P701609), was tag‐free.

Techniques: Knock-Out, Injection, Virus, Western Blot, Immunofluorescence, Expressing

Journal: Advanced Science

Article Title: SIRT3‐Mediated Deacetylation of DRP1 K711 Prevents Mitochondrial Dysfunction in Parkinson's Disease

doi: 10.1002/advs.202411235

Figure Lengend Snippet: Schematic illustration of SIRT3‐mediated DRP1 acetylation is a cause of mitochondrial dysfunction and subsequent damage to dopaminergic neurons in Parkinson's disease.

Article Snippet: The recombinant Sirt3 protein, sourced from MedChemExpress (HY‐P701609), was tag‐free.

Techniques:

Journal: Molecular Neurodegeneration

Article Title: Alpha-synuclein-induced mitochondrial dysfunction is mediated via a sirtuin 3-dependent pathway

doi: 10.1186/s13024-019-0349-x

Figure Lengend Snippet: Antibodies used for western blot and immunocyhistochemistry

Article Snippet: SIRT3 (rabbit) , Novus Biologicals (NBP1–31029) , 1:1000 (WB).

Techniques: Western Blot, Transduction

Journal: Molecular Neurodegeneration

Article Title: Alpha-synuclein-induced mitochondrial dysfunction is mediated via a sirtuin 3-dependent pathway

doi: 10.1186/s13024-019-0349-x

Figure Lengend Snippet: αSyn is found in mitochondria-enriched fraction of H4 SL1&SL2 cells and induces a decrease in SIRT3 expression. a Representative cropped western blots showing αsyn and SIRT3 in cytosolic and mitochondrial fractions at different time points (0 – 72 h) ( b ) αsyn oligomers are apparent after 24 h by luciferase assay (RLU: relative luminescence units), n = 5. Quantification of SIRT3 protein level in mitochondria demonstrates significant decrease in SIRT3 at 48 h and 72 h. c Whole cells lysates from H4 SL1&SL2 cells demonstrate decreased SIRT3 expression after transfection with SIRT3 siRNA n = 6 ( d ) Luciferase activity from αsyn oligonmerization is significantly increased in cells transfected with SIRT3 siRNA (siSIRT3) compared to control siRNA (siCtrl). Error bars represent the mean ± SD. * p < 0.05, ** p < 0.01. Note: In ( a ) αsyn and SIRT3 bands are from different experiments run on different gels. COXIV, GAPDH, and SIRT3 are all from same samples and immunoblot. Loading controls for αsyn blot are not shown. In panel ( c ) αsyn, SIRT3, and GAPDH are detected on same immunoblot

Article Snippet: SIRT3 (rabbit) , Novus Biologicals (NBP1–31029) , 1:1000 (WB).

Techniques: Expressing, Western Blot, Luciferase, Transfection, Activity Assay, Control

Journal: Molecular Neurodegeneration

Article Title: Alpha-synuclein-induced mitochondrial dysfunction is mediated via a sirtuin 3-dependent pathway

doi: 10.1186/s13024-019-0349-x

Figure Lengend Snippet: AICAR activates AMPK-CREB signaling pathway and increases SIRT3 activity and reduces αsyn oligomers. a Representative cropped western blots showing AMPKα, p-AMPKα (Thr 172), CREB, and p-CREB (Ser 133) in H4 SL1&SL2 cells with/without 2 mM AICAR. b SIRT3 expression increases with AICAR-treatment, n = 3. c Luciferase assay ( n = 5) demonstrates AICAR significantly decreases αsyn oligomers and knockdown of SIRT3 (siSIRT3) prevents reduction of αsyn oligomers by AICAR. d Native-page shows AICAR significantly decreases αsyn oligomers. e Representative cropped western blot showing increased Ac-SOD2 (acetyl K68) with no change in total SOD2 in whole cells lysates. AICAR restored acetylated SOD2 levels, n = 3. Error bars represent the mean ± SD, ( n = 3–5). * p < 0.05, ** p < 0.01. In panel ( a ) the same samples were run on different gels and probed separately for AMPKα, p-AMPKα, and GAPDH, and CREB, p-CREB, and GAPDH respectively. In panels ( b ) and ( e ) separate blots were probed for SIRT3 and GAPDH, and COXIV, or SOD2, Ac-SOD2, and GAPDH

Article Snippet: SIRT3 (rabbit) , Novus Biologicals (NBP1–31029) , 1:1000 (WB).

Techniques: Activity Assay, Western Blot, Expressing, Luciferase, Knockdown, Clear Native PAGE

Journal: Molecular Neurodegeneration

Article Title: Alpha-synuclein-induced mitochondrial dysfunction is mediated via a sirtuin 3-dependent pathway

doi: 10.1186/s13024-019-0349-x

Figure Lengend Snippet: Activation of SIRT3 by AICAR attenuates ROS production. a Fluorescence microscopy images of MitoSox-Red and Mitotracker-Green staining in fixed H4 SL1&SL2 cells. MitoSox-Red fluorescence increases when αsyn is overexpressed and AICAR treatment attenuates mtROS. Representative images from 3 experiments. MitoTracker-Green (mitochondria; green); MitoSox-Red (mitochondria; red); merged images (yellow). Scale bar = 10 μm. b Relative intensity of MitoTracker-Green signal between conditions. Quantification of MitoSOX-Red signal intensities ( n = 5), and mean intensity of MitoSOX-Red normalized to MitoTracker-Green intensity ( c ) Representative cropped western blot of HO-1 and GAPDH in whole cells lysates from H4 SL1&SL2 cells. HO-1 level increases at 72 h and is reduced after AICAR-treatment, n = 5. Error bars represent the mean ± SD. * p < 0.05, ** p < 0.01

Article Snippet: SIRT3 (rabbit) , Novus Biologicals (NBP1–31029) , 1:1000 (WB).

Techniques: Activation Assay, Fluorescence, Microscopy, Staining, Western Blot

Journal: Molecular Neurodegeneration

Article Title: Alpha-synuclein-induced mitochondrial dysfunction is mediated via a sirtuin 3-dependent pathway

doi: 10.1186/s13024-019-0349-x

Figure Lengend Snippet: αSyn expression decreases SIRT3 and alters mitochondrial dynamics in vivo. a αSyn oligomers were quantified via luciferase assay in rat brain homogenates at 4 weeks after stereotaxic injection of AAV8-SL1&SL2 in SN, n = 5. b Representative cropped western blots showing αsyn, SIRT3, DRP1 in cytosol and mitochondria from SN of rats 4 weeks after stereotaxic injection of AAV8-SL1 and AAV8-SL2. (C). c Quantification of αsyn, SIRT3, DRP1, protein levels in cytosol and mitochondria from two separate blots for each of 4–5 rats. All bands were normalized to respective loading controls GAPDH and COXIV. d Representative cropped western blot of αsyn, DRP1, and p-DRP1 (Ser 616) in SN lysate of AAV8-SL1&SL2 injected rat. αSyn expression leads to increased p-DRP1 protein levels in ipsilateral (I) injected SN compared to contralateral (C) uninjected SN, n = 5 rats total. In panel ( a ) the same samples were run on one blot that was cropped prior to immunoblotting for αsyn, SIRT3, COXIV, DRP1, and GAPDH. Error bars represent the mean ± SD, ( n = 4–5 rats). * p < 0.05, ** p < 0.01

Article Snippet: SIRT3 (rabbit) , Novus Biologicals (NBP1–31029) , 1:1000 (WB).

Techniques: Expressing, In Vivo, Luciferase, Injection, Western Blot

Journal: Molecular Neurodegeneration

Article Title: Alpha-synuclein-induced mitochondrial dysfunction is mediated via a sirtuin 3-dependent pathway

doi: 10.1186/s13024-019-0349-x

Figure Lengend Snippet: SIRT3 is decreased in human post-mortem brain of neuropathologically confirmed Lewy body disease individuals. a Representative cropped western blot from n = 3 showing decreased SIRT3 in total brain lysates from ten LBD brains compared to ten age-matched healthy controls. b Quantification of αsyn, SIRT3 protein levels from n = 3 western blots. c , d Representative western blot of cytosolic fraction. DRP1 and αsyn were quantified using GAPDH as a loading control. e , f Representative western blot of mitochondrial fraction. DRP1 and αsyn were quantified using COXIV as a loading control. Error bars represent the mean ± SD. ** p < 0.01

Article Snippet: SIRT3 (rabbit) , Novus Biologicals (NBP1–31029) , 1:1000 (WB).

Techniques: Western Blot, Control

Journal: Molecular Neurodegeneration

Article Title: Alpha-synuclein-induced mitochondrial dysfunction is mediated via a sirtuin 3-dependent pathway

doi: 10.1186/s13024-019-0349-x

Figure Lengend Snippet: Cartoon representing of αsyn-induced effect on SIRT3 and mitochondrial dysfunction. Consequences of decreased SIRT3 include decreased AMPK-CREB signaling, impairment in mitochondrial bioenergetics and dynamics, and increased acetylation of SIRT3 substrates such as SOD2 all of which contribute to increased ROS production and neurodegeneration. Question mark indicates pathway not supported by data in this manuscript

Article Snippet: SIRT3 (rabbit) , Novus Biologicals (NBP1–31029) , 1:1000 (WB).

Techniques:

Journal: Journal of Pineal Research

Article Title: Melatonin ameliorates PM 2.5 ‐induced cardiac perivascular fibrosis through regulating mitochondrial redox homeostasis

doi: 10.1111/jpi.12686

Figure Lengend Snippet: Melatonin alleviated PM 2.5 ‐induced cardiac mitochondrial oxidative damage. A, The level of 3′‐NT. B, The level of 4‐HNE. C, The level of GSH/GSSG. D, Representative pictures of myocardial tissue by transmission electron microscope. The blow panels are high magnification (scale bar: 0.2 μm) images corresponding to the upper panels (scale bar: 0.5 μm). E, MitoSOX staining of heart tissues (scale bar: 100 μm). F, Mean fluorescence intensities of heart. G, Representative Western blot pictures. H, The quantitative analysis of SIRT3 protein levels. I, The activity of SIRT3. J, The quantitative analysis of SOD2 acetylation. K, The activity of SOD2. Data are expressed as the means ± SD; n = 6 in each group. * P < .05

Article Snippet: The deacetylase activity of SIRT3 was measured by a SIRT3 Fluorimetric Activity Assay Kit (BPS bioscience) following the manufacturer's protocol.

Techniques: Transmission Assay, Microscopy, Staining, Fluorescence, Western Blot, Activity Assay

Journal: Journal of Pineal Research

Article Title: Melatonin ameliorates PM 2.5 ‐induced cardiac perivascular fibrosis through regulating mitochondrial redox homeostasis

doi: 10.1111/jpi.12686

Figure Lengend Snippet: PM 2.5 regulated cardiac myofibroblast conversion via increased mitochondrial reactive oxygen levels and SOD2 acetylation, decreased SIRT3 expression and activity. A, Cardiac fibroblast cell viability after PM 2.5 ‐ treated. B, Quantification analysis of fluorescence intensity obtained from flow cytometry. C, Representative images of cardiac fibroblast α‐SMA and Vimentin immunostaining. D, Representative Western blot pictures. E, The quantitative analysis of collagen‐I, collagen‐III, and α‐SMA protein levels. F, The quantitative analysis of SIRT3 protein levels. G, The activity of SIRT3. H, The quantitative analysis of SOD2 acetylation. I, The activity of SOD2. Data are expressed as the means ± SD from three independent experiments. * P < 0.05 compared to control group

Article Snippet: The deacetylase activity of SIRT3 was measured by a SIRT3 Fluorimetric Activity Assay Kit (BPS bioscience) following the manufacturer's protocol.

Techniques: Expressing, Activity Assay, Fluorescence, Flow Cytometry, Immunostaining, Western Blot

Journal: Journal of Pineal Research

Article Title: Melatonin ameliorates PM 2.5 ‐induced cardiac perivascular fibrosis through regulating mitochondrial redox homeostasis

doi: 10.1111/jpi.12686

Figure Lengend Snippet: Mitochondrial‐derived ROS mediated PM 2.5 ‐induced cardiac myofibroblast conversion. A, Quantification analysis of fluorescence intensity obtained from flow cytometry. B, The quantitative analysis of α‐SMA protein levels. C, Representative Western blot pictures. D, The quantitative analysis of SIRT3 protein levels. E, The activity of SIRT3. F, The quantitative analysis of SOD2 acetylation. G, The activity of SOD2. Data are expressed as the means ± SD from three independent experiments. * P < .05 compared to control group; # P < .05 compared to PM 2.5 ‐treated group

Article Snippet: The deacetylase activity of SIRT3 was measured by a SIRT3 Fluorimetric Activity Assay Kit (BPS bioscience) following the manufacturer's protocol.

Techniques: Derivative Assay, Fluorescence, Flow Cytometry, Western Blot, Activity Assay

Journal: Journal of Pineal Research

Article Title: Melatonin ameliorates PM 2.5 ‐induced cardiac perivascular fibrosis through regulating mitochondrial redox homeostasis

doi: 10.1111/jpi.12686

Figure Lengend Snippet: Melatonin alleviated the oxidative stress, SIRT3 impairment after PM 2.5 treatment in vitro. A, Cardiac fibroblast cell viability after melatonin ‐ treated. B, Quantification analysis of fluorescence intensity obtained from flow cytometry. C, The quantitative analysis of α‐SMA protein levels. D, Representative Western blot pictures. E, The quantitative analysis of SIRT3 protein levels. F, The activity of SIRT3. G, The quantitative analysis of SOD2 acetylation. H, The activity of SOD2. Data are expressed as the means ± SD from three independent experiments. * P < .05 compared to control group; # P < .05 compared to PM 2.5 ‐treated group

Article Snippet: The deacetylase activity of SIRT3 was measured by a SIRT3 Fluorimetric Activity Assay Kit (BPS bioscience) following the manufacturer's protocol.

Techniques: In Vitro, Fluorescence, Flow Cytometry, Western Blot, Activity Assay

Journal: Journal of Pineal Research

Article Title: Melatonin ameliorates PM 2.5 ‐induced cardiac perivascular fibrosis through regulating mitochondrial redox homeostasis

doi: 10.1111/jpi.12686

Figure Lengend Snippet: 3‐TYP pretreatment abolished the melatonin‐suppressed cardiac myofibroblast conversion induced by PM 2.5 treatment. A, Quantification analysis of fluorescence intensity obtained from flow cytometry. B, The quantitative analysis of α‐SMA protein levels. C, Representative Western blot pictures. D, The quantitative analysis of SIRT3 protein levels. E, The activity of SIRT3. F, The quantitative analysis of SOD2 acetylation. G, The activity of SOD2. Data are expressed as the means ± SD from three independent experiments. * P < .05 compared to PM 2.5 ‐treated group; # P < .05 compared to melatonin pretreatment group; ∆ P < .05 compared to 3‐TYP pretreatment group

Article Snippet: The deacetylase activity of SIRT3 was measured by a SIRT3 Fluorimetric Activity Assay Kit (BPS bioscience) following the manufacturer's protocol.

Techniques: Fluorescence, Flow Cytometry, Western Blot, Activity Assay

Journal: Journal of Cell Science

Article Title: Ethanol sensitizes mitochondria to the permeability transition by inhibiting deacetylation of cyclophilin-D mediated by sirtuin-3

doi: 10.1242/jcs.073502

Figure Lengend Snippet: Ethanol exposure stimulates the peptidyl-prolyl cis-trans isomerase activity of cyclophilin-D and sensitizes the mitochondria to the MPT. (A) H4IIEC3 cells were either left untreated or exposed to 25 mM of ethanol in the absence or presence of 5 mM 4-MP. Following 24 or 48 hours of incubation, the cells were harvested and mitochondria isolated. Alternatively, cells were transfected with siRNA targeting CyP-A or CyP-D. The western blot on the left shows mitochondrial extracts that were assessed for cyclophilin-D activity and cyclophilin-D or A expression. The graph on the right shows the quantification of these experiments; values are the means from triplicate samples, and the error bars indicate standard deviations. P<0.05 for control versus ethanol and ethanol versus ethanol+4-MP by one way ANOVA and Scheffe's post-hoc test. (B) H4IIEC3 cells were either left untreated or transfected with 50 nM of a non-target siRNA or an siRNA targeting sirtuin-3. After 24 hours, cells were either left untreated or exposed to 25 mM ethanol in the absence or presence of 4-MP. After 48 hours, the cells were harvested and the mitochondria isolated. Mitochondrial respiration was initiated by the addition of 1 mM malate and 1 mM glutamate. To trigger mitochondrial swelling, 50 μM Ca2+ was added at time points indicated. The change in absorbance was measured spectrophotometrically.

Article Snippet: Measurement of sirtuin-3 and cyclophilin-D activity Sirtuin-3 activity was measured in mitochondrial extracts by using the Cyclex sirtuin-3 assay kit (MBL).

Techniques: Activity Assay, Incubation, Isolation, Transfection, Western Blot, Expressing

Journal: Journal of Cell Science

Article Title: Ethanol sensitizes mitochondria to the permeability transition by inhibiting deacetylation of cyclophilin-D mediated by sirtuin-3

doi: 10.1242/jcs.073502

Figure Lengend Snippet: Ethanol exposure inhibits sirtuin-3 activity and promotes cyclophilin-D acetylation and binding to the adenine nucleotide translocator-1. (A) H4IIEC3 cells were left untreated or exposed to 25 mM of ethanol for 24 or 48 hours in the absence or presence of 4-MP. The cells were harvested and the NAD+:NADH and sirtuin-3 activity was determined in whole-cell and mitochondrial extracts, respectively. The values are the means from triplicate samples, and the error bars indicate standard deviations. P<0.05 for control versus ethanol and ethanol versus ethanol+4-MP by one-way ANOVA and Scheffe's post-hoc test. (B) H4IIEC3 cells were left untreated or exposed to 25 mM of ethanol for 24 or 48 hours in the absence or presence of 4-MP. The cells were harvested and the mitochondria isolated. Sirtuin-3 expression was determined by western blotting. Acetyl-CoA sythetase 2 (AceCS2) was immunoprecipitated from mitochondrial extracts. The western blots of the immunoprecipitates were probed with antibody against acetylated lysine, stripped and then re-probed with antibody against AceCS2. (C) H4IIEC3 cells were left untreated or exposed to 25 mM of ethanol for 24 or 48 hours in the absence or presence of 4-MP. The cells were harvested and the mitochondria isolated. Cyclophilin-D was immunoprecipitated from mitochondrial extracts. The western blots of the immunoprecipitates were probed with antibody against acetylated lysine, stripped and then re-probed with antibody against cyclophilin-D. (D) H4IIEC3 cells were either left untreated or exposed to 25 mM of ethanol for 24 or 48 hours. The cells were harvested and mitochondria isolated. ANT-1 was immunoprecipitated from mitochondrial extracts. The western blots of the immunoprecipitates were probed with antibodies against cyclophilin-D or ANT-1. To access cyclophilin-D acetylation, the blots were stripped and then re-probed with antibody against acetylated lysine.

Article Snippet: Measurement of sirtuin-3 and cyclophilin-D activity Sirtuin-3 activity was measured in mitochondrial extracts by using the Cyclex sirtuin-3 assay kit (MBL).

Techniques: Activity Assay, Binding Assay, Isolation, Expressing, Western Blot, Immunoprecipitation

Journal: Journal of Cell Science

Article Title: Ethanol sensitizes mitochondria to the permeability transition by inhibiting deacetylation of cyclophilin-D mediated by sirtuin-3

doi: 10.1242/jcs.073502

Figure Lengend Snippet: Suppression of sirtuin-3 expression recapitulates the effects of ethanol exposure on cyclophilin-D acetylation, activity and binding to the ANT-1. (A) H4IIEC3 cells were transfected with 50 nM siRNA targeting sirtuin-1, 3 or a non-targeting control. Following 48 hours of incubation, the cells were harvested and mitochondria isolated. Mitochondrial extracts were separated by SDS-PAGE and then electroblotted onto PVDF membranes. Western blots were probed with antibody against sirtuin-3 (panel 1). Alternatively, cyclophilin-D was immunoprecipitated from mitochondrial extracts. The immunoprecipitates were separated by SDS-PAGE and electroblotted onto PVDF membranes. The western blots were probed with antibody against acetylated lysine, then stripped and re-probed with an antibody against cyclophilin-D (panels 2 and 3). Cyclophilin-D activity was determined fluorescently in mitochondrial extracts. The values are the means of three samples, and error bars indicate standard deviations. P<0.05 for non-target siRNA versus sirtuin-3 siRNA. (B) H4IIEC3 cells were transfected with 50 nM of siRNA targeting sirtuin-1, 3 or a non-targeting control. Following 48 hours of incubation, cells were harvested and mitochondria isolated. ANT-1 was immunoprecipitated from mitochondrial extracts. The immunoprecipitates were separated by SDS-PAGE and electroblotted onto PVDF membranes. Western blots were then probed with antibodies against cyclophilin-D or ANT-1. To access cyclophilin-D acetylation, the cyclophilin-D blots were stripped and then re-probed with antibody against acetylated lysine. (C) (Left) Western blot of H4IIEC3 cells that were either left untreated or exposed to 25 mM of ethanol for 48 hours. Cyclophilin-D was immunoprecipitated from mitochondrial extracts and incubated with recombinant sirtuins. The immunoprecipitates were then run out on SDS-PAGE gels and electroblotted onto PVDF membranes. Blots were then probed with antibody against acetylated lysine, stripped and re-probed with antibody against cyclophilin-D. (Right) Quantification of cyclophilin-D activity. Cyclophilin-D immunoprecipitates that had been incubated with recombinant sirtuins. Cyclophilin-D activity was determined fluorescently as described in Materials and Methods. Values are the means from triplicate samples, and error bars indicate standard deviations. P<0.05 for control versus ethanol and ethanol versus ethanol+sirtuin-3 by one-way ANOVA and Scheffe's post-hoc test.

Article Snippet: Measurement of sirtuin-3 and cyclophilin-D activity Sirtuin-3 activity was measured in mitochondrial extracts by using the Cyclex sirtuin-3 assay kit (MBL).

Techniques: Expressing, Activity Assay, Binding Assay, Transfection, Incubation, Isolation, SDS Page, Western Blot, Immunoprecipitation, Recombinant

Journal: Journal of Cell Science

Article Title: Ethanol sensitizes mitochondria to the permeability transition by inhibiting deacetylation of cyclophilin-D mediated by sirtuin-3

doi: 10.1242/jcs.073502

Figure Lengend Snippet: Increased cyclophilin-D acetylation and decreased sirtuin-3 activity in mitochondria isolated from ethanol-fed rats and mouse hepatocytes exposed to ethanol. (A) Western blots of mitochondria that had been isolated from the liver of control or ethanol-fed rats. Cyclophilin-D was immunoprecipitated from mitochondrial extracts. The immunoprecipitates were separated by SDS-PAGE and electroblotted onto PVDF membranes. Blots were probed with antibody against acetylated lysine, then stripped and re-probed with an antibody against cyclophilin-D. Alternatively, mitochondrial extracts were used to determine the expression of sirtuin-3 by using anti-sirtuin-3 antibody. (B) Quantification of cyclophilin-D and sirtuin-3 activity. Mitochondria that had been isolated from the livers of control or ethanol-fed rats. Cyclophilin-D or sirtuin-3 activity was determined fluorescently in mitochondrial extracts. The values are the means from triplicate samples, and the error bars indicate standard deviations. P<0.05 for control versus ethanol. (C) (Left) Western blots (left) of mouse hepatocytes that had been left untreated or were transfected with siRNA targeting sirtuin-3 or exposed to ethanol. Following 48 hours, the hepatocytes were harvested and mitochondria isolated. Cyclophilin-D was immunoprecipitated from mitochondrial extracts. The immunoprecipitates were then separated by SDS-PAGE and electroblotted. Blots were then probed with antibody against acetylated lysine, stripped and re-probed with antibody against cyclophilin-D. (Right) Quantification of sirtuin-3 and cyclophilin-D activity. Mouse hepatocytes were untreated or exposed to ethanol for 48 hours in the absence or presence of 4-MP. Sirtuin-3 or cyclophilin-D activity was determined fluorescently in mitochondrial extracts as described in Materials and Methods. The values are the means from triplicate samples, and the error bars indicate standard deviations. P<0.05 for control versus ethanol and ethanol versus ethanol+4-MP by one-way ANOVA and Scheffe's post-hoc test. (D) (Left) Mouse hepatocytes were transfected with 50 nM of siRNA targeting sirtuin-3, cyclophilin-D or a non-targeting control; 24 hours after transfection, cells were left untreated or exposed to 25 mM of ethanol for 48 hours. The cells were then harvested and mitochondria isolated. Where shown (arrows), 50 μM Ca2+ was added. The change in absorbance was measured spectrophotometrically at 540 nm. (Right) At 24 hours after transfection, mouse hepatocytes were left untreated or exposed to 25 mM of ethanol for 48 hours. Cells were then treated with 10 ng/ml of TNF. At the times indicated, the viability of the cells was assessed. Values are the means of three samples, and the error bars indicate standard deviations. P<0.05 for TNF(control) versus TNF(ethanol), TNF(ethanol) versus TNF(ethanol-siCyP-D) and TNF(control) versus TNF(siSirt-3) by one-way ANOVA and Scheffe's post-hoc test.

Article Snippet: Measurement of sirtuin-3 and cyclophilin-D activity Sirtuin-3 activity was measured in mitochondrial extracts by using the Cyclex sirtuin-3 assay kit (MBL).

Techniques: Activity Assay, Isolation, Western Blot, Immunoprecipitation, SDS Page, Expressing, Transfection

Journal: Journal of Cell Science

Article Title: Ethanol sensitizes mitochondria to the permeability transition by inhibiting deacetylation of cyclophilin-D mediated by sirtuin-3

doi: 10.1242/jcs.073502

Figure Lengend Snippet: Activation of AMPK by AICAR reverses the inhibitory effect of ethanol exposure on sirtuin-3 activity. (A) (Left) Western blots of H4IIEC3 cells that had been either left untreated or exposed to 25 mM of ethanol for 48 hours. Where indicated, cells had been treated with 0.5 mM of AICAR for another 8 hours. The cells were harvested and cell extracts were separated by SDS-PAGE and electroblotted onto PVDF membranes. Blots were probed with antibodies against AMPK or specific for AMPK phosphorylated on Thr172. (Right) Quantification of the NAD+:NADH ratio. Cell extracts were used to determine the NAD+:NADH ratio fluorescently as described in Materials and Methods. Values are the means of three samples, error bars indicate standard deviations. P<0.05 for control versus ethanol and ethanol versus ethanol+AICAR by one-way ANOVA and Scheffe's post-hoc test. (B) Quantification of AMPK activity. H4IIEC3 cells were either left untreated or exposed to 25 mM of ethanol for 48 hours. Where indicated cells were subsequently treated with 0.5 mM of AICAR. At the time points indicated the cells were harvested and AMPK activity was determined. The values are the means of three samples, error bars indicate standard deviations. P<0.05 for control versus ethanol, control versus control+AICAR and ethanol versus ethanol+AICAR by one-way ANOVA and Scheffe's post-hoc test. (C) Quantification of sirtuin-3 activity. H4IIEC3 cells were either left untreated or exposed to 25 mM of ethanol for 48 hours. Where indicated, cells were treated with 0.5 mM of AICAR. At the time points indicated, the cells were harvested and mitochondria isolated. Sirtuin-3 activity was measured in mitochondrial extracts. Values are the means of three samples, error bars indicate standard deviations. P<0.05 for control versus ethanol and ethanol versus ethanol+AICAR by one-way ANOVA and Scheffe's post-hoc test.

Article Snippet: Measurement of sirtuin-3 and cyclophilin-D activity Sirtuin-3 activity was measured in mitochondrial extracts by using the Cyclex sirtuin-3 assay kit (MBL).

Techniques: Activation Assay, Activity Assay, Western Blot, SDS Page, Isolation

Journal: Journal of Cell Science

Article Title: Ethanol sensitizes mitochondria to the permeability transition by inhibiting deacetylation of cyclophilin-D mediated by sirtuin-3

doi: 10.1242/jcs.073502

Figure Lengend Snippet: Sirtuin-3 is necessary for AICAR to reverse the ethanol-induced activation, acetylation and binding of cyclophilin-D to ANT-1. (A) Western blots of H4IIEC3 cells that had been transfected with non-targeting siRNA or siRNA against sirtuin-3. After 24 hours, the cells were either left untreated or exposed to 25 mM of ethanol for 48 hours. Following ethanol exposure, were indicated, the cells were treated with 0.5 mM of AICAR for 8 hours. The mitochondria were then isolated and mitochondrial extracts were immunoprecipitated with cyclophilin-D antibody. The immunoprecipitates were separated by SDS-PAGE and electroblotted onto PVDF membranes. Blots were probed with antibody against acetylated lysine, then stripped and re-probed with an antibody against cyclophilin-D. (B) Western blots of H4IIEC3 cells that had been transfected with non-targeting siRNA or siRNA against sirtuin-3. After 24 hours, the cells were either left untreated or exposed to 25 mM of ethanol for 48 hours. Where indicated, cells were subsequently treated with 0.5 mM of AICAR for 8 hours. Mitochondrial extracts were immunoprecipitated with antibody against ANT-1. The immunoprecipitates were separated by SDS-PAGE and electroblotted onto PVDF membranes. Blots were then probed with antibodies against cyclophilin-D or ANT-1. To access cyclophilin-D acetylation, the cyclophilin-D blots were stripped and then re-probed with antibody against acetylated lysine. (C) Quantification of cyclophilin-D activity. H4IIEC3 cells were transfected with non-target control siRNA or siRNA against sirtuin-3. After 24 hours, the cells were either left untreated or exposed to 25 mM of ethanol for 48 hours. Subsequently, where indicated, the cells were treated with 0.5 mM of AICAR. At the times indicated, cells were harvested and mitochondria isolated. Cyclophilin-D activity was measured in mitochondrial extracts as described in Materials and Methods. The values are the means of three samples, error bars indicate standard deviations. P<0.05 for control versus ethanol, ethanol versus ethanol+AICAR siN.T. and ethanol+AICAR siN.T. versus ethanol+AICAR siSirt-3 by one-way ANOVA and Scheffe's post-hoc test.

Article Snippet: Measurement of sirtuin-3 and cyclophilin-D activity Sirtuin-3 activity was measured in mitochondrial extracts by using the Cyclex sirtuin-3 assay kit (MBL).

Techniques: Activation Assay, Binding Assay, Western Blot, Transfection, Isolation, Immunoprecipitation, SDS Page, Activity Assay

Journal: Journal of Cell Science

Article Title: Ethanol sensitizes mitochondria to the permeability transition by inhibiting deacetylation of cyclophilin-D mediated by sirtuin-3

doi: 10.1242/jcs.073502

Figure Lengend Snippet: Sirtuin-3 is required for AMPK activation to reverse the ethanol-induced sensitization to onset of the MPT and TNF-induced cytotoxicity. (A) H4IIEC3 cells were transfected with non-target control siRNA or siRNA against sirtuin-3. After 24 hours, the cells were left untreated or exposed to 25 mM of ethanol for 48 hours. Where indicated, the cells were subsequently treated with 0.5 mM of AICAR for 8 hours. Mitochondria were isolated. Where shown, 50 μM of Ca2+ was added. The change in absorbance was measured at 540 nm. (B) H4IIEC3 cells were transfected with non-target control siRNA or siRNA against sirtuin-3. After 24 hours, the cells were left untreated or exposed to 25 mM of ethanol for 48 hours. Where indicated, the cells were subsequently treated with 0.5 mM of AICAR for 8 hours. Cells were then treated with 10 ng/ml of TNF. At the times indicated, the viability of the cells was assessed. The values are the means of three samples, and error bars indicate standard deviations. P<0.05 for control(+TNF) versus ethanol(+TNF), ethanol(+TNF) versus ethanol(+TNF+AICAR), ethanol(+TNF+AICAR) versus ethanol(+TNF+AICAR)siSirt-3 and control(+TNF) versus control(+TNF)siSirt-3 by one-way ANOVA and Scheffe's post-hoc test.

Article Snippet: Measurement of sirtuin-3 and cyclophilin-D activity Sirtuin-3 activity was measured in mitochondrial extracts by using the Cyclex sirtuin-3 assay kit (MBL).

Techniques: Activation Assay, Transfection, Isolation

Journal: Journal of Cell Science

Article Title: Ethanol sensitizes mitochondria to the permeability transition by inhibiting deacetylation of cyclophilin-D mediated by sirtuin-3

doi: 10.1242/jcs.073502

Figure Lengend Snippet: Acetylation of the Lys145 residue in cyclophilin-D controls sensitivity to PTP induction and TNF cytotoxicity in ethanol-exposed cells. (A) H4IIEC3 cells expressing CyP-D(K145R) or CyP-D(K145Q) were generated. Cells were then either left untreated or exposed to 25 mM of ethanol for 48 hours. Mitochondria were then isolated. Mitochondrial respiration was initiated by the addition of 1 mM malate and 1 mM glutamate. To trigger mitochondrial swelling, 50 μM Ca2+ was added as indicated. The change in absorbance was measured at 540 nm. (B) (Left) H4IIEC3 cells stably expressing CyP-D(K145R) or CyP-D(K145Q) were exposed to 25 mM of ethanol for 48 hours. Cells were then treated with 10 ng/ml TNF. Alternatively, cells expressing CyP-D(K145R) were transfected with siRNA against sirtuin-3. Following 48 hours, the cells were treated with TNF. (Right) Cells were transfected with siRNA targeting sirt-3 and Cyp-D. Following 48 hours, cells were treated with 10 ng/ml TNF. At the times indicated, the viability of the cells was assessed. The values are the means of three samples, and error bars indicate standard deviations. P<0.05 for control+TNF versus ethanol+TNF, ethanol+TNF versus ethanol+TNF(CyP-DK145R), control+TNF versus TNF(CyP-DK145Q) and TNF(siSirt-3) versus TNF(siSirt-3,CyP-DK145R), TNF(siSirt-3) versus TNF(siSirt-3, siCyP-D) by one-way ANOVA and Scheffe's post-hoc test.

Article Snippet: Measurement of sirtuin-3 and cyclophilin-D activity Sirtuin-3 activity was measured in mitochondrial extracts by using the Cyclex sirtuin-3 assay kit (MBL).

Techniques: Expressing, Generated, Isolation, Stable Transfection, Transfection

Journal: Journal of Cell Science

Article Title: Ethanol sensitizes mitochondria to the permeability transition by inhibiting deacetylation of cyclophilin-D mediated by sirtuin-3

doi: 10.1242/jcs.073502

Figure Lengend Snippet: Maintenance of the NAD+:NADH ratio prevents the ethanol-induced decline of sirtuin-3 activity. (A) (Left) H4IIEC3 cells were untreated or exposed to 25 mM of ethanol in the absence or presence of 10 mM of acetoacetate (AcA) for 48 hours. Cell extracts were prepared to determine the NAD+:NADH ratio. (Right) Western blots of H4IIEC3 cells treated as above. Cyclophilin-D was immunoprecipitated from mitochondrial extracts. The immunoprecipitates were then separated by SDS-PAGE and electroblotted onto PVDF membranes. Blots were then probed with antibody against cyclophilin-D, stripped and reprobed with antibody against acetylated lysine. The values are the means of three samples, and the error bars indicate standard deviations. P<0.05 for control versus ethanol, ethanol versus ACA and ethanol versus ethanol+AcA by one-way ANOVA and Scheffe's post-hoc test. (B) H4IIEC3 cells were untreated or exposed to 25 mM of ethanol in the absence or presence of 10 mM of AcA. After 24 or 48 hours incubation, cyclophilin-D or sirtuin-3 activity was determined in mitochondrial extracts as described in Materials and Methods. The values are the means of three samples, error bars indicate standard deviations. P<0.05 for control versus ethanol, ethanol versus AcA and ethanol versus ethanol+AcA by one-way ANOVA and Scheffe's post-hoc test. (C) (Left) H4IIEC3 cells were untreated or exposed to 25 mM of ethanol in the absence or presence of 10 mM AcA. Following a 48-hour incubation, mitochondria were isolated. Where shown (arrows), a 50 μM Ca2+ was added. (Right) H4IIEC3 cells were treated with 10 ng/ml TNF. At the times indicated, the viability of the cells was assessed. The values are the means of three samples and the error bars indicate standard deviations. P<0.05 for control(+TNF) versus ethanol(+TNF) and ethanol(+TNF) versus ethanol+TNF+AcA by one-way ANOVA and Scheffe's post-hoc test.

Article Snippet: Measurement of sirtuin-3 and cyclophilin-D activity Sirtuin-3 activity was measured in mitochondrial extracts by using the Cyclex sirtuin-3 assay kit (MBL).

Techniques: Activity Assay, Western Blot, Immunoprecipitation, SDS Page, Incubation, Isolation

Journal: Journal of Cell Science

Article Title: Ethanol sensitizes mitochondria to the permeability transition by inhibiting deacetylation of cyclophilin-D mediated by sirtuin-3

doi: 10.1242/jcs.073502

Figure Lengend Snippet: Ethanol-induced decline of sirtuin-3 activity sensitizes mitochondria to MPT induction by TNF. Ethanol metabolism induces a decline of sirtuin-3 activity, causing an increase in cyclophilin-D acetylation and activity, which results in a sensitization to induction of the permeability transition by TNF.

Article Snippet: Measurement of sirtuin-3 and cyclophilin-D activity Sirtuin-3 activity was measured in mitochondrial extracts by using the Cyclex sirtuin-3 assay kit (MBL).

Techniques: Activity Assay, Permeability