Journal: Cardiovascular Research

Article Title: Sirtuin-1 directly binds and deacetylates hepatic PCSK9 thereby promoting the inhibition of LDL receptor degradation

doi: 10.1093/cvr/cvaf087

Figure Lengend Snippet: Exogenous SIRT1 treatment reduces plasma LDL-cholesterol and protects against atherosclerosis in ApoE −/− mice. Eight-week-old ApoE −/− mice were fed on a high cholesterol diet (1.25% w/w) for 4 weeks and randomized to be treated with rmSIRT1 ( n = 6) or vehicle (PBS containing 0.1% BSA) ( n = 6) for another 4 weeks. ( A ) Plasma concentration of SIRT1 measured using ELISA of C57BL/6J wild-type mice ( n = 6), ApoE −/− mice + vehicle ( n = 6) and ApoE −/− mice + rmSIRT1 ( n = 6). ( B ) Weekly measurements of body weight of ApoE −/− mice + vehicle ( n = 6) and ApoE −/− mice + rmSIRT1 ( n = 6). ( C ) Graph showing cholesterol distribution in the different lipoprotein sub-fractions separated by gel filtration chromatography; ( D ) Bar graph of plasma total cholesterol and ( E ) LDL-cholesterol concentrations. ( F ) Representative pictures (left) and quantifications of ( G ) thoracic-abdominal aortae en face , ( H ) aortic root cross sections stained with ORO and ( I ) immunohistochemically for macrophages (CD68). Scale bars in photomicrographs: 1 mm (for E ) and 500 μm (for F , G ). Grey bars represent vehicle treatment and green bars represent rmSIRT1 treatment. ORO, Oil-Red O. Values are represented as means ± SEM. Statistical significance was performed using Student’s unpaired t -test.

Article Snippet: Where indicated, cells were treated with vehicle control (Phosphate buffer saline containing 0.1% BSA) or recombinant human SIRT1 (rhSIRT1, 1 μmol/L) (CSB-EP822202HU, Cusabio Biotech, LubioScience, Switzerland) or recombinant human PCSK9 (rhPCSK9, ab198471, Abcam, concentrations in Figure legends) for 2 h.

Techniques: Clinical Proteomics, Concentration Assay, Enzyme-linked Immunosorbent Assay, Filtration, Chromatography, Staining

Journal: Cardiovascular Research

Article Title: Sirtuin-1 directly binds and deacetylates hepatic PCSK9 thereby promoting the inhibition of LDL receptor degradation

doi: 10.1093/cvr/cvaf087

Figure Lengend Snippet: Exogenous SIRT1 increases hepatic LDLR by post-translational modification of PCSK9 in ApoE −/− mice. Eight-week-old ApoE −/− mice were fed on a high cholesterol diet (1.25% w/w) for 4 weeks and were randomized to be treated with rmSIRT1 ( n = 6) or with vehicle (PBS with 0.1% BSA) ( n = 6) for another 4 weeks. ( A ) Western blot of LDLR (120 kDa) and PCSK9 (∼62 kDa) in hepatic tissue lysates of mice ( n = 5). β-actin (45 kDa) was used as loading control. Bar graph of ELISA of ( B ) total PCSK9 and ( C ) acetylated PCSK9 in mice plasma. Statistical significance was performed using Student’s unpaired two-sample t -test. ( D , E , F ) Human hepatoma Huh7 cells [three independent triplicate experiments ( n = 3)] were treated with either 1 μmol/L of recombinant human SIRT1 (rhSIRT1) or vehicle (PBS containing 0.1% BSA) for 2 h at 37°C. The cell culture media was collected, and cells were lysed to perform ELISA to detect PCSK9. Bar graph of ELISA of ( D ) total cellular PCSK9, ( E ) total media PCSK9 and ( F ) acetylated PCSK9 in the media of cultured Huh7 cells. Grey bars represent vehicle treatment and green bars represent rmSIRT1 or rhSIRT1 treatment as indicated. Data are represented as means ± SEM. Statistical significance was performed using Student’s unpaired t -test.

Article Snippet: Where indicated, cells were treated with vehicle control (Phosphate buffer saline containing 0.1% BSA) or recombinant human SIRT1 (rhSIRT1, 1 μmol/L) (CSB-EP822202HU, Cusabio Biotech, LubioScience, Switzerland) or recombinant human PCSK9 (rhPCSK9, ab198471, Abcam, concentrations in Figure legends) for 2 h.

Techniques: Modification, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Recombinant, Cell Culture

Journal: Cardiovascular Research

Article Title: Sirtuin-1 directly binds and deacetylates hepatic PCSK9 thereby promoting the inhibition of LDL receptor degradation

doi: 10.1093/cvr/cvaf087

Figure Lengend Snippet: Exogenous SIRT1 inhibits PCSK9-mediated LDLR degradation in hepatocytes. HuH-7 (hereafter Huh7) cells were transfected with a specific siRNA against LDLR or with non-silencing control siRNA (NS control). Assays were performed 72 h post transfection. Cells were then pre-treated with 1 μmol/L of rhSIRT1 for 2 h ( A ) Specific cellular binding of 125 I-LDL was measured at 4°C ( B ) Specific cellular association of 125 I-LDL was measured at 37°C. ( C , D , E ) Huh7 cells were co-incubated with rhPCSK9 (2 μg/mL) with rhSIRT1 (1 μmol/L) for 2 h at 37°C ( C ) Representative Western blots ( n = 3) and quantification of blot density of LDLR (130 kDa) degradation in Huh7 cells. Vinculin (140 kDa) was used as the loading control. ( D ) Specific cellular binding of 125 I-LDL was measured at 4°C ( E ) Specific cellular association of 125 I-LDL was measured at 37°C. Values are represented as means ± SEM of three independent triplicate or more experiments ( n = 3). Statistical significance was performed using one-way ANOVA followed by Tukey’s multiple comparison test.

Article Snippet: Where indicated, cells were treated with vehicle control (Phosphate buffer saline containing 0.1% BSA) or recombinant human SIRT1 (rhSIRT1, 1 μmol/L) (CSB-EP822202HU, Cusabio Biotech, LubioScience, Switzerland) or recombinant human PCSK9 (rhPCSK9, ab198471, Abcam, concentrations in Figure legends) for 2 h.

Techniques: Transfection, Control, Binding Assay, Incubation, Western Blot, Comparison

Journal: Cardiovascular Research

Article Title: Sirtuin-1 directly binds and deacetylates hepatic PCSK9 thereby promoting the inhibition of LDL receptor degradation

doi: 10.1093/cvr/cvaf087

Figure Lengend Snippet: Exogenous SIRT1 inhibits PCSK9 activity through direct deacetylation in hepatocytes—specific mutation of these deacetylation sites restores PCSK9 activity. Huh7 cells were incubated with rhSIRT1 (1 μmol/L) for 2 h at 37°C. ( A ) Western blot analysis of PCSK9 immunoprecipitated Huh7 cells lysates with total PCSK9 antibody (∼62 kDa) and acetyl lysine antibody (Ac-K-PCSK9, ∼62 kDa). ( B ) Direct binding assay interaction between rhSIRT1 and rhPCSK9 using Surface Plasmon Resonance. The equilibrium dissociation constant was 34 nM. ( C ) Schematic diagram of PCSK9 domains and an overview of lysine(K) sites on PCSK9 deacetylated by SIRT1. Representative Western blot and quantification of LDLR (130 kDa) and Vinculin (140 kDa) expression in Huh7 cells expressing WT, 3KR, 3KQ mutants of PCSK9 for 72 h ( n = 3). (E )V. means empty vector ( D ) Specific cellular binding of 125 I-LDL was measured at 4°C ( E ) Specific cellular association of 125 I-LDL was measured at 37°C. ( F ) Binding of acetylated and deacetylated PCSK9 to EGF-A of LDLR using ELISA. SP—signal peptide, Pro—pro-domain, Catalytic—catalytic domain, CHRD—cysteine, histidine-rich domain. Values are represented as means ± SEM of three independent triplicate or more experiments ( n = 3). Statistical significance was performed using one-way ANOVA followed by Tukey’s multiple comparison test.

Article Snippet: Where indicated, cells were treated with vehicle control (Phosphate buffer saline containing 0.1% BSA) or recombinant human SIRT1 (rhSIRT1, 1 μmol/L) (CSB-EP822202HU, Cusabio Biotech, LubioScience, Switzerland) or recombinant human PCSK9 (rhPCSK9, ab198471, Abcam, concentrations in Figure legends) for 2 h.

Techniques: Activity Assay, Mutagenesis, Incubation, Western Blot, Immunoprecipitation, Binding Assay, SPR Assay, Expressing, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Journal of Clinical Investigation

Article Title: The ZIP8/SIRT1 axis regulates alveolar progenitor cell renewal in aging and idiopathic pulmonary fibrosis

doi: 10.1172/jci157338

Figure Lengend Snippet: Figure 3. ZIP8-dependent zinc metabolism regulates AEC2 renewal through SIRT1. (A) IPA pathway analysis of AEC2s from healthy and IPF lungs.(B) Sir- tuin activation score of healthy and IPF AEC2s. (C and D) SIRT1 expression in freshly isolated AEC2s (C) and AEC2s derived from 3D organoids (D) by qPCR (n = 4–6, *P < 0.05). (E) SIRT1 expression of AEC2s derived from 3D-cultured organoids of ZIP8+ and ZIP8– AEC2s (n = 5 each, *P < 0.05). (F) Intracellular SIRT1 levels in healthy and IPF AEC2s without and with ZnSO4 treatment by flow cytometry (n = 3–4). (G) Intracellular SIRT1 levels in gated ZIP8+ and ZIP8– AEC2s from healthy lungs (n = 4). (H and I) CFE with 3D organoid culture of AEC2s from healthy lungs treated with either 1 μM SRT1720 (n = 4–5, ***P < 0.001 by ANOVA) (H) or 125 and 200 μM splitomicin (n = 3, ***P < 0.001, ****P < 0.0001 by ANOVA) (I). (J) CFE of AEC2s from IPF lungs cultured with SRT1720 at the indicated concentrations (n = 3, **P < 0.01, ****P < 0.0001 by ANOVA). (K) CFE of AEC2s from IPF lungs cultured with 1 μM SRT1720, 100 μM ZnSO4, or both (n = 3–4, ****P < 0.0001 by ANOVA). (L–N) A549 cells with SIRT1 knockout and control cells. SIRT1 ko 1, set 1 sgRNA; SIRT ko 2, set 2 sgRNA. (L) SIRT1 expression by qPCR. (M) SIRT1 expression by Western blot analysis; the same experiments were performed 3 times. (N) CFE with 3D organoid culture (n = 4, ***P < 0.001 by ANOVA). Data are shown as mean ± SEM. Unpaired 2-tailed Student’s t test.

Article Snippet: Human SIRT1 monoclonal mouse IgG1 (clone 834918, catalog IC7714S) from R&D Systems was used for intracellular staining.

Techniques: Activation Assay, Expressing, Isolation, Derivative Assay, Cell Culture, Flow Cytometry, Knock-Out, Control, Western Blot

Journal: Journal of Clinical Investigation

Article Title: The ZIP8/SIRT1 axis regulates alveolar progenitor cell renewal in aging and idiopathic pulmonary fibrosis

doi: 10.1172/jci157338

Figure Lengend Snippet: Figure 4. Downregulated ZIP8/SIRT1 signaling and decreased renewal capacity of AEC2s from old mouse lungs. (A) Flow cytometry analysis to gate out total AEC2s (R2), and ZIP8 expression AEC2s (R3) from lung homologies of young and old mice. (B) Percentage of ZIP8+ cells (R3) within total AEC2s (n = 5–6, **P < 0.01). (C) Number of ZIP8+ cells recovered from young and old mouse lung (n = 5–6, **P < 0.01). (D) CFE of mouse AEC2s isolated from young and old mouse lungs (n = 6–7, **P < 0.01). (E and F) Flow cytometry analysis of ZIP8 expression in gated AEC2s cultured with medium only or medium containing 100 μM ZnSO4 (n = 6, ***P < 0.001). (G–J) 3D organoid culture of AEC2s isolated from lungs of 2.5-, 12-, 14-, and 18-month-old mice with and without 100 μM ZnSO4 treatment. (G) CFE (n = 3–4, *P < 0.05, ***P < 0.001, ****P < 0.0001 by ANOVA). (H–J) Expression of Slc39a8 (H), Sftpc (I), and Pdpn (J) in AEC2s derived from 3D-cultured organoids with and without ZnSO4 treatment by qPCR (n = 3–4, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by ANOVA). (K) Violin plots of gene expression in AEC2s from lungs of bleomycin-treated young and old mice. (L) IPA pathway analysis of AEC2s from young and old mice on day 4 after bleomycin injury. (M and N) CFE of AEC2s from uninjured 10- to 12-week-old young mice treated with SRT1720 (n = 3–6, **P < 0.01, ****P < 0.0001 by ANOVA) (M) and splitomicin (n = 5–6, ****P < 0.0001 by ANOVA) (N) at the indicated doses and DMSO control. (O) CFE of AEC2s from uninjured 20- to 24-month-old mice treated with SRT1720 at the indicated doses and DMSO control (n = 4, **P < 0.01, ****P < 0.0001 by ANOVA). Data are shown as mean ± SEM. Unpaired 2-tailed Student’s t test.

Article Snippet: Human SIRT1 monoclonal mouse IgG1 (clone 834918, catalog IC7714S) from R&D Systems was used for intracellular staining.

Techniques: Flow Cytometry, Expressing, Isolation, Cell Culture, Derivative Assay, Gene Expression, Control

Journal: Journal of Clinical Investigation

Article Title: The ZIP8/SIRT1 axis regulates alveolar progenitor cell renewal in aging and idiopathic pulmonary fibrosis

doi: 10.1172/jci157338

Figure Lengend Snippet: Figure 5. Targeted deletion of Slc39a8 decreased AEC2 renewal. (A) ZIP-expressing cells among gated AEC2s and (B) percentage of ZIP8+ cells within the total AEC2 population from uninjured Zip8AEC2 and control mice by flow cytometry (n = 8, ****P < 0.001). (C and D) Intracellular zinc levels of AEC2s (C) and percentage of zinc+ AEC2s within the total AEC2 population (D) from Zip8AEC2 (n = 4) and control mice (n = 8) by flow cytometry (**P < 0.01). (E) CFE of flow-sorted AEC2s from uninjured Zip8AEC2 and control mice with 3D organoid culture (n = 6 each, *P < 0.05). (F and G) Expression of Sirt1 (n = 4, *P < 0.05) (F) and Pdpn (n = 5, **P < 0.01) (G) in AEC2s derived from 3D-cultured organoids. (H–J) AEC2s from day 4 bleomycin-injured Zip8AEC2 and control mice. (H) Number of AEC2s recovered per lung (n = 5, ***P < 0.001). (I and J) CFE of AEC2s with 3D organoid culture (n = 5–7, **P < 0.01) (I) and colony size (n = 28–86, ****P < 0.0001) (J). (K and L) Ki-67 expression by flow cytometry (n = 6 each, *P < 0.05) (K) and Pdpn expression by qPCR (n = 5 each, ****P < 0.0001) (L) in AEC2s derived from 3D-cultured organoids. (M and N) Violin plots of gene expression in AEC2s with scRNA-Seq. (M) AEC2s from 2-month-old (Young) and 18- to 20-month-old (Old) C57BL/6 WT mice (n = 3). (N) AEC2s from 10- to 12-week-old (young) Zip8AEC2 mice and littermate controls 2 weeks after 4 doses of tamoxifen injection. Data are shown as mean ± SEM. Unpaired 2-tailed Student’s t test.

Article Snippet: Human SIRT1 monoclonal mouse IgG1 (clone 834918, catalog IC7714S) from R&D Systems was used for intracellular staining.

Techniques: Expressing, Control, Flow Cytometry, Derivative Assay, Cell Culture, Gene Expression, Injection

Journal: Journal of Clinical Investigation

Article Title: The ZIP8/SIRT1 axis regulates alveolar progenitor cell renewal in aging and idiopathic pulmonary fibrosis

doi: 10.1172/jci157338

Figure Lengend Snippet: Figure 7. Summary of the role of the ZIP8/SIRT1 axis in regulating alveolar progenitor cell renewal. In young and healthy AEC2s, sufficient ZIP8 ensures adequate levels of intracellular zinc, SIRT1 activity, and AEC2 renewal capacity. However, in old AEC2s and IPF AEC2s, severely downregulated ZIP8 results in intracellular zinc deficiency and defective SIRT1 activity, which impairs AEC2 renewal. In addition, enzymes regulating NAD+ synthesis were downregulated in IPF AEC2s, further exaggerating SIRT1 impairment. Therefore, the optimal combinations of zinc, NAD+, and SIRT1 activation may restore AEC2 integrity and mitigate fibrosis. MT, metallothionein.

Article Snippet: Human SIRT1 monoclonal mouse IgG1 (clone 834918, catalog IC7714S) from R&D Systems was used for intracellular staining.

Techniques: Activity Assay, Activation Assay

Journal: Oxidative Medicine and Cellular Longevity

Article Title: SIRT1 Activity in Peripheral Blood Mononuclear Cells Correlates with Altered Lung Function in Patients with Chronic Obstructive Pulmonary Disease

doi: 10.1155/2018/9391261

Figure Lengend Snippet: SIRT1 protein expression (a) and activity (b) in peripheral blood mononuclear cells (PBMCs). Data are expressed as mean ± SD. Sirtuin 1 (SIRT1) expression and activity were determined in the nuclei extracted from PBMCs of healthy nonsmokers (HnS), healthy smokers (HS), and COPD patients (COPD), respectively indicated with black circle, black square, and black triangle.

Article Snippet: SIRT1 expression was measured by enzyme-linked immunosorbent assay (SIRT1 ELISA Kit, Elabscience), following the manufacturer's instructions.

Techniques: Expressing, Activity Assay

Journal: Oxidative Medicine and Cellular Longevity

Article Title: SIRT1 Activity in Peripheral Blood Mononuclear Cells Correlates with Altered Lung Function in Patients with Chronic Obstructive Pulmonary Disease

doi: 10.1155/2018/9391261

Figure Lengend Snippet: Linear regression analysis between Sirtuin 1 (SIRT1) activity in peripheral blood mononuclear cells (PBMCs) and forced expiratory volume in 1 second (FEV1) values (a); between SIRT1 activity in peripheral blood mononuclear cells (PBMCs) and forced expiratory volume in 1 second/forced vital capacity (FEV1/FVC) ratio values (b); and between SIRT1 activity in peripheral blood mononuclear cells (PBMCs) and serum TEAC (c). SIRT1 activity was determined in the nuclei extracted from PBMCs of healthy nonsmokers (HnS), healthy smokers (HS), and COPD patients (COPD). The multivariate analysis was also adjusted for age, gender, BMI, smoking pack/years, comorbidity, and drugs.

Article Snippet: SIRT1 expression was measured by enzyme-linked immunosorbent assay (SIRT1 ELISA Kit, Elabscience), following the manufacturer's instructions.

Techniques: Activity Assay