Journal: Biofactors (Oxford, England)

Article Title: Comparative effects of viable Lactobacillus rhamnosus GG and its heat‐inactivated paraprobiotic in the prevention of high‐fat high‐fructose diet‐induced non‐alcoholic fatty liver disease in rats

doi: 10.1002/biof.2116

Figure Lengend Snippet: Schematic representation of the mechanisms of action regulated by probiotic or paraprobiotic administration in the prevention of diet‐induced hepatic lipid accumulation. ALT: alanine aminotransferase; AST: aspartate aminotransferase; FAS: fatty‐acid synthase; FATP2: fatty‐acid transport protein 2; HFHF: high‐fat high‐fructose; LPL: lipoprotein lipase; MTTP: microsomal triglyceride transfer protein; SIRT3: NAD‐dependent deacetylase sirtuin‐3; PARA: paraprobiotic; PRO: probiotic; SREBP‐1c: sterol regulatory element‐binding protein 1; TG: triglyceride; VLDL: very‐low‐density lipoprotein.

Article Snippet: Protein levels were detected via specific antibodies for FATP2 (1:1000), AQP9 (1:1000), SIRT3 (1:1000) and TFAM (1:1000) (SantaCruz Biotech, Dallas, TX, USA); CD36 (1:1000), AMPK (1:1000), ATGL (1:1000), HSL (1:1000), β‐actin (1:1000) and α‐tubulin (1:1000) (Cell Signaling Technology, Danvers, MA, USA); DGAT2 (1:1000), SCD1 (1:1000), SIRT1 (1:1000) and SREBP‐1c (1:1000) (Abcam, Cambridge, UK); PGC‐1α (1:1000) (BioTechne, Minneapolis, Minnesota, USA).

Techniques: Histone Deacetylase Assay, Binding Assay

Journal: Cell death & disease

Article Title: Oroxylin A inhibits glycolysis-dependent proliferation of human breast cancer via promoting SIRT3-mediated SOD2 transcription and HIF1α destabilization.

doi: 10.1038/cddis.2015.86

Figure Lengend Snippet: Figure 1 OA inhibits glycolysis by upregulating SIRT3 and SIRT3-mediated destabilization of HIF1α. (a) MDA-MB-231 cells were treated with OA under conditions of hypoxia or normoxia for 10 h. Glucose uptake was measured using the Amplex Red assay, and production of lactic acid was assayed with the Lactic Acid production Detection kit. (b) SIRT3-deficient MDA-MB-231 cells were incubated with 100 μM OA for 10 h under hypoxia. Glucose uptake and production of lactic acid were assayed as above. (c, d) The protein expressions (c) and mRNA expressions (d) of SIRT3, HIF1α and HK II was detected by immunoblotting and quantitative RT-PCR, respectively, in cells treated with OA for 10 h under hypoxia. (e, f) SIRT3-deficient cells were treated with/without OA for 10 h under hypoxia. The protein expressions of SIRT3, HIF1α and HK II (e) and the mRNA expression of HK II (f) were detected. (g–i) HIF1α overexpressed MDA-MB-231 cells were then incubated with 100 μM OA for 10 h under conditions of hypoxia. Glucose uptake and production of lactic acid (g), the protein expression (h) and the mRNA expression (i) of HK II were assayed. Bars, S.D.; *Po0.05 or **Po0.01 versus untreated controls in hypoxia

Article Snippet: The HIF1α human cDNA clone and SIRT3 human cDNA clone were obtained from OriGene (OriGene Technologies, Inc., Rockville, MD, USA).

Techniques: Amplex Red Assay, Incubation, Western Blot, Quantitative RT-PCR, Expressing

Journal: Cell death & disease

Article Title: Oroxylin A inhibits glycolysis-dependent proliferation of human breast cancer via promoting SIRT3-mediated SOD2 transcription and HIF1α destabilization.

doi: 10.1038/cddis.2015.86

Figure Lengend Snippet: Figure 2 OA destabilized HIF1α by influencing SIRT3-modulated activity of PDH. (a) MDA-MB-231 cells were treated with NAM (10 mM) for 10 h under hypoxic conditions. HIF1α was immunoprecipitated using SIRT3 or SIRT1 antibodies. Western blot assays were performed for HIF1α, SIRT1 and SIRT3. (b) For 10 h OA treatment under hypoxia, the expression of PHD was detected by immunoblotting. (c) MDA-MB-231 cells were treated with OA for 10 h under hypoxic or normoxia conditions. The expression of hydroxylated HIF1α (HIF-OH) was detected by immunoblotting. (d) MDA-MB-231 cells treated with or without 100 μM OA or 3 μM MG-132 or 1 mM DMOG for 10 h as indicated were immunoblotted against antibodies specific to HIF-OH, or total HIF1α. (e) SIRT3-deficient MDA-MB-231 cells were incubated with 100 μM OA for 10 h under hypoxia. HIF-OH was detected by immunoblotting. (f) SIRT3-deficient MDA-MB-231 cells were incubated with or without 100 μM OA or 1 mM DMOG for 10 h under hypoxia. Hydroxylated HIF1α (HIF-OH) and total HIF1α were detected by immunoblotting

Article Snippet: The HIF1α human cDNA clone and SIRT3 human cDNA clone were obtained from OriGene (OriGene Technologies, Inc., Rockville, MD, USA).

Techniques: Activity Assay, Immunoprecipitation, Western Blot, Expressing, Incubation

Journal: Cell death & disease

Article Title: Oroxylin A inhibits glycolysis-dependent proliferation of human breast cancer via promoting SIRT3-mediated SOD2 transcription and HIF1α destabilization.

doi: 10.1038/cddis.2015.86

Figure Lengend Snippet: Figure 3 OA-induced activation of PDHs through the regulation of ·O2 −levels by SIRT3. (a) MDA-MB-231 cells were treated with OA for 10 h under hypoxia. The levels of cellular superoxide anion (·O2 −) and hydrogen peroxide (H2O2) were detected by FACSCalibur flow cytometry using the fluorescent dye dihydroethidium at Ex/Em of 300/610 nm or dichlorofluorescein-diacetate at Ex/Em of 488/525 nm, respectively. (b) SIRT3-deficient MDA-MB-231 cells were incubated with or without 100 μM OA for 10 h under hypoxia, and ·O2 −was detected. (c, d) Cells were treated with 100 μM OA or 10 mM N-acetyl cysteine (antioxidant used as a positive control) in the presence of 1 μM rotenone for 10 h under hypoxia. The levels of ·O2 −(c), protein expression of HIF-OH and HIF1α (d) were detected. (e, f) Cells were treated with 100 μM OA with/without 1 μM rotenone for 10 h under hypoxia. Cell survival rate (e), glucose uptake and production of lactic acid (f) were detected, respectively. Bars, S.D.; *Po0.05 or **Po0.01 versus untreated control

Article Snippet: The HIF1α human cDNA clone and SIRT3 human cDNA clone were obtained from OriGene (OriGene Technologies, Inc., Rockville, MD, USA).

Techniques: Activation Assay, Flow Cytometry, Incubation, Positive Control, Expressing, Control

Journal: Cell death & disease

Article Title: Oroxylin A inhibits glycolysis-dependent proliferation of human breast cancer via promoting SIRT3-mediated SOD2 transcription and HIF1α destabilization.

doi: 10.1038/cddis.2015.86

Figure Lengend Snippet: Figure 4 The regulations of OA on glycolysis and HIF1α were associated with increased SOD2 levels under hypoxia. (a–c) MDA-MB-231 cells were treated with OA for 10 h under hypoxia. The protein expression (a), mRNA expression (b) and activity (c) of SOD2 were detected by immunoblotting, Cu/Zn-SOD and Mn-SOD Assay Kit, and quantitative RT-PCR, respectively. (d, e) SIRT3-deficient MDA-MB-231 cells were incubated with or without 100 μM OA for 10 h under hypoxia conditions. The protein expression (d) and activity (e) of SOD2 were detected as described above. (f, g) SOD2-deficient MDA-MB-231 cells were incubated with or without 100 μM OA for 10 h under hypoxia. Glucose uptake and production of lactic acid (f), and protein expressions of HIF-OH, HIF1α, and HKII (g) were detected. (h) MDA-MB-231 cells were treated with OA for 10 h under hypoxia. SOD2 was immunoprecipitated using SIRT3 antibody, and acetylated-SOD2 (Ac-SOD2) was immunoprecipitated using SOD2 antibody in mitochondrial extracts. (i–k) MDA-MB-231 cells were treated with or without 100 μM OA or 2 μM TAS or 10 mM NAM for 10 h under hypoxia. (i) Ac-SOD2 was immunoprecipitated using SOD2 antibody in mitochondrial extracts. (j) The activity of SOD2 was detected. (k) The protein expressions of SOD2, HIF-OH, HIF1α, and HK II was detected by immunoblotting. (l, m) SIRT3- deficient MDA-MB-231 cells were incubated with or without 100 μM OA or 10 mM NAM for 10 h under hypoxia. Ac-SOD2 was immunoprecipitated in mitochondrial extracts (l), and the activity of SOD2 (m) was assayed. Bars, S.D.; *Po0.05 or **Po0.01 versus untreated controls

Article Snippet: The HIF1α human cDNA clone and SIRT3 human cDNA clone were obtained from OriGene (OriGene Technologies, Inc., Rockville, MD, USA).

Techniques: Expressing, Activity Assay, Western Blot, Quantitative RT-PCR, Incubation, Immunoprecipitation

Journal: Cell death & disease

Article Title: Oroxylin A inhibits glycolysis-dependent proliferation of human breast cancer via promoting SIRT3-mediated SOD2 transcription and HIF1α destabilization.

doi: 10.1038/cddis.2015.86

Figure Lengend Snippet: Figure 5 OA regulated the mRNA expression of SOD2 through the interaction of SIRT3 with FOXO3a in mitochondria. (a–d) MDA-MB-231 cells were treated with OA for 10 h under hypoxia conditions. (a) Mitochondria were isolated after treatment and subjected to western blot analysis for FOXO3a. (b) FOXO3a was immunoprecipitated using a SIRT3 antibody. (c) ChIP analysis of FOXO3a binding to the SOD2 promoter in MDA-MB-231 cells treated with OA. Cells were fixed with 1% formaldehyde to crosslink protein–DNA interactions, sonicated, and fixed cells were immunoprecipitated with anti-FOXO3a antibody. DNA was eluted and purified before analysis using specific primers and quantitative RT- PCR. (d) Nuclei were isolated after treatment and subjected to EMSA to assess the binding of FOXO3a to the SOD2 promoter in mitochondria. (e) SIRT3-deficient and normal MDA- MB-231 cells were treated with OA or 10 mM NAM for 10 h under hypoxia conditions. SOD2 promoter luciferase reporter plasmid (p3x-FOXO3a-luc) was transfected into MDA-MB-231 cells. Luciferase activity was normalized to Renilla activity and expressed as luciferase/Renilla relative units. Bars, S.D.; *Po0.05 or **Po0.01 versus untreated controls

Article Snippet: The HIF1α human cDNA clone and SIRT3 human cDNA clone were obtained from OriGene (OriGene Technologies, Inc., Rockville, MD, USA).

Techniques: Expressing, Isolation, Western Blot, Immunoprecipitation, Binding Assay, Sonication, Purification, Quantitative RT-PCR, Luciferase, Plasmid Preparation, Transfection, Activity Assay

Journal: The Journal of Cell Biology

Article Title: Drosophila Sirt2/mammalian SIRT3 deacetylates ATP synthase β and regulates complex V activity

doi: 10.1083/jcb.201404118

Figure Lengend Snippet: Human ATP synthase β is an acetylated protein, and its deacetylation is regulated by SIRT3. (A) pCMV vector or ATP synthase β (DDK tagged) was transfected in HEK293T cells, immunoprecipitated using an antibody to DDK tag, and probed with an antibody to acetyl-Lys (Ac-Lys). (B) HEK293T cells were cotransfected with ATP synthase β (ATP syn β) and either SIRT3 siRNA or scrambled siRNA. ATP synthase β was immunoprecipitated, and its acetylation status was assessed. The bottom blot shows reduction of SIRT3 protein upon siRNA treatment. Knockdown of SIRT3 increases acetylation of ATP synthase β. (C) Expression vector for wild-type SIRT3 was cotransfected in HEK293T cells with ATP synthase β, and its acetylation status was assessed after immunoprecipitation. Overexpression of SIRT3 decreases acetylation of ATP synthase β. (D) HEK293T cells were cotransfected with ATP synthase β and either SIRT4 siRNA or scrambled siRNA. SIRT4 knockdown does not affect acetylation of ATP synthase β. (E) Wild-type SIRT4 expression vector was cotransfected in HEK293T cells with ATP synthase β, and its acetylation status was assessed after immunoprecipitation. SIRT4 overexpression does not affect acetylation of ATP synthase β. (F) HEK293T cells were cotransfected with ATP synthase β and either SIRT5 siRNA or scrambled siRNA. SIRT5 knockdown does not affect acetylation of ATP synthase β. (G) Wild-type SIRT5 expression vector was cotransfected in HEK293T cells with ATP synthase β, and its acetylation status was assessed after immunoprecipitation. SIRT5 overexpression does not affect acetylation of ATP synthase β. (H) HEK293T cells were cotransfected with ATP synthase β and either SIRT1 siRNA or scrambled siRNA. SIRT1 knockdown does not affect acetylation of ATP synthase β. (I) Wild-type SIRT1 expression vector was cotransfected in HEK293T cells with ATP synthase β, and its acetylation status was assessed after immunoprecipitation. SIRT1 overexpression does not affect acetylation of ATP synthase β. (J) Mitochondria were prepared from SIRT3 siRNA–treated or scrambled siRNA–treated cells, and complex V activity was measured. The activity of mitochondria from scrambled siRNA treatment was taken as 100%. SIRT3 knockdown results in an ∼40% decrease in complex V activity. n = 3; error bars represent SDs. **, P ≤ 0.01–0.001 in Student’s t test. (K) Endogenous ATP synthase β was immunoprecipitated from HEK293T cells overexpressing SIRT3, and the immunoprecipitate was probed with antibodies to ATP synthase β and SIRT3. SIRT3 can coimmunoprecipitate with ATP synthase β. IP, immunoprecipitation; WB, Western blot.

Article Snippet: DDK-tagged (similar to FLAG tag) ATP synthase β (RC201638) and DDK-tagged human SIRT3 (RC200190), SIRT4 (RC212226), SIRT5 (RC200189), and SIRT1 (RC218134) plasmids were obtained from OriGene.

Techniques: Plasmid Preparation, Transfection, Immunoprecipitation, Knockdown, Expressing, Over Expression, Activity Assay, Western Blot

Journal: The Journal of Cell Biology

Article Title: Drosophila Sirt2/mammalian SIRT3 deacetylates ATP synthase β and regulates complex V activity

doi: 10.1083/jcb.201404118

Figure Lengend Snippet: Acetylation of ATP synthase β at Lys 259 and Lys 480 regulates complex V activity. (A) Nondegradable (non-deg) ATP synthase β (ATP syn β) is resistant to targeted siRNA-mediated degradation. (B) siRNA-resistant versions of ATP synthase β wherein Lys 259 or Lys 480 either individually or together were substituted with Arg or Gln and cotransfected with siRNA to ATP synthase β. Mitochondria were prepared, and complex V activity was measured using an immunocapture assay followed by the amount of ATP synthase β in the same samples. The activity of siRNA-resistant ATP synthase β is taken as 100%. Substitution of either Lys or both with Arg results in increased activity, whereas substitution with Gln results in decreased complex V activity. **, P ≤ 0.01–0.001; ***, P ≤ 0.001–0.0001. (C) An overview of the crystal structure of bovine mitochondrial F1–stator complex is shown on the left in ribbon representation. The F1 domain contains 3α (green), 3β (purple), and a single subunit of γ (pink). The stator complex shows portions of subunit b (teal), oligomycin sensitivity-conferring protein (orange), and F6 (yellowish green). The right shows a closer view of the region around the active site (marked by the black box in the left image). The Lys residues are shown as spheres, and the active site amino acids are shown as stick models. Acetylation of Lys 259 (Lys 206 in the crystal structure) and Lys 480 (430 in the crystal structure) could affect protein conformation near the active site. (D) Endogenous ATP synthase β was immunoprecipitated from human breast cancer cell lines, and its acetylation status was assessed using an acetyl-Lys antibody. ATP synthase β is more acetylated in MDA-MB-231 cells compared with T47D. (E) Complex V activity was measured in mitochondria prepared from human breast cancer cell lines. The activity is significantly less in MDA-MB-231 cells compared with that in T47D cells. n = 3. Analysis of variance was performed, and Tukey’s honest significance test was applied to determine significance. T47D–MDA-MB-231: adjusted P = 1.0 × 10 −7 ; T47D–MDA-MB-435: adjusted P = 1.9 × 10 −5 . (F) Oxygen consumption is less in MDA-MB-231 compared with that in T47D mitochondria. n = 3. Analysis of variance was performed, and Tukey’s honest significance test was applied to determine significance. T47D–MDA-MB-231: adjusted P = 2.0 × 10 −6 ; T47D–MDA-MB-435: adjusted P = 1.0 × 10 −5 . (G) A model depicting Drosophila Sirt2/mammalian SIRT3-mediated deacetylation of ATP synthase β and its impact on complex V activity. Error bars represent SDs. IP, immunoprecipitation; WB, Western blot.

Article Snippet: DDK-tagged (similar to FLAG tag) ATP synthase β (RC201638) and DDK-tagged human SIRT3 (RC200190), SIRT4 (RC212226), SIRT5 (RC200189), and SIRT1 (RC218134) plasmids were obtained from OriGene.

Techniques: Activity Assay, Immunoprecipitation, Western Blot

Journal: Cellular signalling

Article Title: Adiponectin enhances the bioenergetics of cardiac myocytes via an AMPK- and succinate dehydrogenase-dependent mechanism.

doi: 10.1016/j.cellsig.2020.109866

Figure Lengend Snippet: Fig. 5. Adiponectin potentiates cellular bioenergetics via a Sirt3-dependent mechanism - Cultured cardiac myocytes were treated with 10–20 moi of adenoviral vectors harboring either a control shRNA (Control, 0.01 μg/ml Adiponectin, and 250 μM AICAR) or one targeting Sirt3 (si-Sirt3), as indicated. a-d. After 24 h, the cells remained in normoxic conditions or e-h. were exposed to hypoxia, in DMEM containing 17.5 mM glucose plus 100 μM palmitate-BSA, for an additional 24 h. a-h. Following this period, cells were treated with either 0.01 μg/ml Adiponectin for 1 h or 250 μM AICAR for 24 h. OCR was then measured in live cells using an extracellular flux analyzer (XFe96), before and after sequentially treating the cells with oligomycin (oligo), FCCP, and antimycin A and rotenone (AA/Rtn), where indicated by the arrows shown in (a.). a, e. The curves represent the real-time measurements of OCR (pmole/min, Y-axis), v. time (min, X-axis). b, f. The bar graphs represent mitochondrial basal OCR and SRC, and c, g. the proton leak, and ATP production levels, as explained in the legend of Fig. 1. b, h. Plots of OCR (pmole/min, Y-axis) v. ECAR (mpH/min, X-axis) represent the energy phenotypes of the cells under the different conditions. The open boxes represent the basal (unstressed values of OCR and ECAR), the closed boxes represent the maximum (stressed values of OCR and ECAR), connected by dashed lines. The color code key for the different samples is shown on of the top of each curve. Bar graphs represent average values of OCR (n = 8) for the different parameter described, indicated above, error bars represent S.E.M., and *p ≤0.05 v. control, #p ≤0.05 v. si-Sirt3-treated.

Article Snippet: The human cDNA clones for Sdhaf1 (NM_001042631) and Sirt3 (NM_012239) were purchased (Origene Technologies, Inc.) and cloned into recombinant, replication-defective, adenovirus serotype 5 (Ad5), as we previously described [24].

Techniques: Cell Culture, Control, shRNA

Journal: Cellular signalling

Article Title: Adiponectin enhances the bioenergetics of cardiac myocytes via an AMPK- and succinate dehydrogenase-dependent mechanism.

doi: 10.1016/j.cellsig.2020.109866

Figure Lengend Snippet: Fig. 6. SDH assembly is sufficient for potentiating cellular bioenergetics - Cultured cardiac myocytes were treated with 10–20 moi of adenoviral vectors harboring either a control shRNA virus or one targeting Sirt3 (si-Sirt3), as indicated. After 24 h, the cells were the treated with 10–20 moi of the control virus or ones harboring wild type Sdhaf1 (wt-Sdhaf1) or K25R mutant Sdhaf1 (mt-Sdhaf1). a-d. The cells remained in normoxic conditions or e-h. were exposed to hypoxia, in DMEM containing 17.5 mM glucose plus 100 μM palmitate-BSA, for an additional 24 h. OCR was then measured in live cells using an extracellular flux analyzer (XFe96), before and after sequentially treating the cells with oligomycin (oligo), FCCP, and antimycin A and rotenone (AA/Rtn), where indicated by the arrows shown in (a.). a, e. The curves represent the real-time measurements of OCR (pmole/min, Y-axis) v. time (min, X-axis). b, f. The bar graphs represent mitochondrial basal OCR and SRC, and c, g. the proton leak, and ATP production levels, as explained in the legend of Fig. 1. d, g. Plots of OCR (pmole/min, Y-axis) v. ECAR (mpH/min, X-axis) represent the energy phenotypes of the cells under the different conditions. The open boxes represent the basal (unstressed values of OCR and ECAR), the closed boxes represent the maximum (stressed values of OCR and ECAR), connected by dashed lines. The color code key for the different samples is shown on of the top of each curve. Bar graphs represent average values of OCR (n = 8) for the different parameter described, indicated above, error bars represent S.E.M., and *p ≤0.05 v. control, #p ≤0.05 v. si-Sirt3-treated.

Article Snippet: The human cDNA clones for Sdhaf1 (NM_001042631) and Sirt3 (NM_012239) were purchased (Origene Technologies, Inc.) and cloned into recombinant, replication-defective, adenovirus serotype 5 (Ad5), as we previously described [24].

Techniques: Cell Culture, Control, shRNA, Virus, Mutagenesis

Journal: Cellular signalling

Article Title: Adiponectin enhances the bioenergetics of cardiac myocytes via an AMPK- and succinate dehydrogenase-dependent mechanism.

doi: 10.1016/j.cellsig.2020.109866

Figure Lengend Snippet: Fig. 7. Overexpression of Sdhaf1 is sufficient for promoting SDH assembly in a Sirt3-dependent manner - Cultured cardiac myocytes were treated with 10–20 moi of adenoviral vectors harboring either a control shRNA (Control, wt- Sdhaf1, mt-Sdhaf1), or one targeting Sirt3 (siSirt3), as indicated. After 24 h, the cells were then treated with 10–20 moi of the control virus or ones harboring wild type Sdhaf1 (wt-Sdhaf1) or Arg-25 mutant Sdhaf1 (mt-Sdhaf1). The cells then remained in normoxic conditions or were exposed to hypoxia, in DMEM containing 17.5 mM glucose plus 100 μM palmitate-BSA, for an addi tional 24 h. Subsequently, mitochondrial protein was extracted and analyzed by non-denaturing (native) gel electrophoresis and Western blotting. a. Images of the native gel after electrophoresis and before membrane transfer of proteins (lower image), after Western blot analysis with anti-Sdhb (top) or anti-Sha (middle). b. The Western blot signals of Sdhb, for each condition (n = 3–4), were quantified and normalized to the band observed on the native gel image (a. boxed). Bar graphs represent average of these values relative to the control adjusted to 1, error bars represent S.E.M., and *p ≤0.05 v. control normoxia, or #p ≤0.05 v. control hypoxia.

Article Snippet: The human cDNA clones for Sdhaf1 (NM_001042631) and Sirt3 (NM_012239) were purchased (Origene Technologies, Inc.) and cloned into recombinant, replication-defective, adenovirus serotype 5 (Ad5), as we previously described [24].

Techniques: Over Expression, Cell Culture, Control, shRNA, Virus, Mutagenesis, Nucleic Acid Electrophoresis, Western Blot, Electrophoresis, Membrane

Journal: Cellular signalling

Article Title: Adiponectin enhances the bioenergetics of cardiac myocytes via an AMPK- and succinate dehydrogenase-dependent mechanism.

doi: 10.1016/j.cellsig.2020.109866

Figure Lengend Snippet: Fig. 8. A diagram depicting the summary and con clusions of this study. Adiponectin acutely (within 1 h of stimulation) boosts the cardiac myocytes’ bio energetics via activation of AMPK, which through a Sirt3-dependent mechanism that we have not deter mined yet, enhances the assembly of SDH complex (Sdha, Sdhb, Sdhc, Sdhd, depicted in yellow). This, in turn, contributes to the increased activity of the TCA cycle and ETC (complexes I-V, indicated by the nu merals I-V), which augment basal, maximal OCR, and ATP-linked OCR (simulated by the Seahorse analyzer trace of OCR vs. TIME, Fig. 8). Therefore, this has the potential to increase the cells’ tolerance to hypoxia, which dampens all these parameters.

Article Snippet: The human cDNA clones for Sdhaf1 (NM_001042631) and Sirt3 (NM_012239) were purchased (Origene Technologies, Inc.) and cloned into recombinant, replication-defective, adenovirus serotype 5 (Ad5), as we previously described [24].

Techniques: Activation Assay, Activity Assay

Journal: Oncotarget

Article Title: SIRT3 is attenuated in systemic sclerosis skin and lungs, and its pharmacologic activation mitigates organ fibrosis.

doi: 10.18632/oncotarget.12504

Figure Lengend Snippet: Figure 1: SIRT3 negatively regulates fibrotic responses. A.-C. Normal adult lung fibroblasts transfected with SIRT3 or empty vector were incubated with TGF-β2 (10 ng/ml) for 24 h. A. Results of real-time qPCR normalized with GAPDH are means ± SD of triplicate determinations from an experiment representative of three. *p < 0.05. B. Whole cell lysates were examined by Western analysis. Representative blots from an experiment representative of three; -fold change in band intensities normalized with tubulin shown below. Cgn I, Type I collagen. C. Fibroblasts were co-transfected [SBE]4-luc along with SIRT3 or empty vector. Whole cell lysates were analyzed for their luciferase activities. Results are the means ± SD of triplicate experiments. *p < 0.05. D. Fibroblasts were transiently transfected with SIRT3 siRNA or scrambled (control) siRNA and incubated for 48 h. Results of qRT-PCR normalized with GAPDH are means ± SD of triplicate determinations from an experiment representative of three. *p < 0.05.

Article Snippet: Subconfluent cultures were transfected with SIRT3 expression vector (OriGene , Rockville, MD) or empty vector, SBE4-TK-Luc or siRNA (Santa Cruz) for 48 h, followed by incubation with or without TGF-β2 (10 ng/ ml) and hexafluoro for 24 h. Cultures were harvested and whole-cell lysates were assayed for their luciferase activities using the Dual-Luciferase Reporter Assay system (Promega, Madison, WI) [43].

Techniques: Transfection, Plasmid Preparation, Incubation, Western Blot, Luciferase, Control, Quantitative RT-PCR

Journal: Oncotarget

Article Title: SIRT3 is attenuated in systemic sclerosis skin and lungs, and its pharmacologic activation mitigates organ fibrosis.

doi: 10.18632/oncotarget.12504

Figure Lengend Snippet: Figure 2: Hexafluoro stimulates SIRT3 expression. Lung fibroblasts were incubated with hexafluoro (10 µM or indicated concentrations) for 24h or indicated periods, in the absence or presence of TGF-β2 (10 ng/ml and indicated times). A. Whole cell lysates were examined by Western analysis. Representative blots. – fold change in band intensities normalized with tubulin shown below. B., C. Real-time qPCR results normalized with GAPDH are means ±SD of triplicate determinations from an experiment representative of three. *, p < 0.05.

Article Snippet: Subconfluent cultures were transfected with SIRT3 expression vector (OriGene , Rockville, MD) or empty vector, SBE4-TK-Luc or siRNA (Santa Cruz) for 48 h, followed by incubation with or without TGF-β2 (10 ng/ ml) and hexafluoro for 24 h. Cultures were harvested and whole-cell lysates were assayed for their luciferase activities using the Dual-Luciferase Reporter Assay system (Promega, Madison, WI) [43].

Techniques: Expressing, Incubation, Western Blot

Journal: Oncotarget

Article Title: SIRT3 is attenuated in systemic sclerosis skin and lungs, and its pharmacologic activation mitigates organ fibrosis.

doi: 10.18632/oncotarget.12504

Figure Lengend Snippet: Figure 6: Hexafluoro abrogates fibrotic signaling. A., C. Lung fibroblasts were treated with 10 μM hexafluoro in the presence or absence of TGF-β (10ng/ml) for 120 min. A. Cytoplasmic lysates and nuclear extracts were examined by Western analysis. Representative immunoblots. B. NHLF transiently transfected [SBE]4-luc along with SIRT3 or empty vector, were pre-incubated with 10 μM hexafluoro for 30 min, followed with TGF-β (10 ng/ml) for 24 h. Whole cell lysates were analyzed for their luciferase activities. Results are the means ±SD of triplicate experiments. *p < 0.05. C. Whole cell lysates were examined by Western analysis. Representative immunoblots. Cgn I, Type I collagen. D. Confluent SIRT3-null (SIRT3-/-) and wild-type (WT) embryonic fibroblasts (MEFs) in parallel were preincubated with hexafluoro (10 µM) for 30 min, followed by TGF-β (10 ng/ml) for 24 h. Whole cell lysates were examined by Western analysis. Band intensities normalized to tubulin are shown as -fold change below.

Article Snippet: Subconfluent cultures were transfected with SIRT3 expression vector (OriGene , Rockville, MD) or empty vector, SBE4-TK-Luc or siRNA (Santa Cruz) for 48 h, followed by incubation with or without TGF-β2 (10 ng/ ml) and hexafluoro for 24 h. Cultures were harvested and whole-cell lysates were assayed for their luciferase activities using the Dual-Luciferase Reporter Assay system (Promega, Madison, WI) [43].

Techniques: Western Blot, Transfection, Plasmid Preparation, Incubation, Luciferase

Journal: Oncotarget

Article Title: SIRT3 is attenuated in systemic sclerosis skin and lungs, and its pharmacologic activation mitigates organ fibrosis.

doi: 10.18632/oncotarget.12504

Figure Lengend Snippet: Figure 8: Attenuated SIRT3 expression and activity in SSc and in mouse models of fibrosis. A. Immmunohistochemistry of skin biopsies. Healthy control (n = 3) and dcSSc (n = 9) skin biopsies were evaluated using antibodies to SIRT3. Left panels, representative images. Arrowheads indicate immunopositive spindle-shaped cells within the dermis. Lower panels, higher magnification of boxed areas. Bar = 12.5 μm. Right panel, SIRT3 scores calculated as described in Materials and Methods. Bars represent the means ±SD; p < 0.05. B. Immunfluorescence. Left panels, immunostain of healthy control (n = 5) and SSc (n = 20) skin biopsies. Representative images. Green, Ac-MnSOD; red, αSMA; blue, DAPI. White dashed lines indicated epidermis-dermis junction. Arrows, Ac-MnSOD positive cells; star, Ac-MnSOD and αSMA double-positive cells. Bar = 100 μm (upper panels) and 10 μm (lower panels). Right panel, Ac-MnSOD levels. Each dot represents a biopsy. C. Immunohistochemistry of control (n = 3) and SSc (n = 6) lung biopsies using anti-Ac-MnSOD. Representative images. Inset, higher magnification. Arrow, Ac-MnSOD-positive alveolar macrophages; asterisk, Ac-MnSOD-positive stromal cells. Bar = 50 μm. D., E. Real-time qRT-PCR of lesional skin. Results, normalized with GAPDH, and the means ± SD of each group. *p < 0.05. D. RNA from confluent control (n = 4) and SSc (n = 4) skin fibroblasts was subjected to real-time qPCR. E. Mice treated with s.c. bleomycin (BLM; n = 4) or PBS (n = 4) for 14 days in parallel were sacrificed on day 28. RNA isolated from lesional skin was analyzed by qRT-PCR. Results, normalized with Gapdh, are means ± SD of each group. *p < 0.05.

Article Snippet: Subconfluent cultures were transfected with SIRT3 expression vector (OriGene , Rockville, MD) or empty vector, SBE4-TK-Luc or siRNA (Santa Cruz) for 48 h, followed by incubation with or without TGF-β2 (10 ng/ ml) and hexafluoro for 24 h. Cultures were harvested and whole-cell lysates were assayed for their luciferase activities using the Dual-Luciferase Reporter Assay system (Promega, Madison, WI) [43].

Techniques: Expressing, Activity Assay, Control, Immunohistochemistry, Quantitative RT-PCR, Isolation