Journal: Cancer Cell

Article Title: Discovery, In Vivo Activity, and Mechanism of Action of a Small-Molecule p53 Activator

doi: 10.1016/j.ccr.2008.03.004

Figure Lengend Snippet: SirT1-Related Effects of Tenovins in Mammalian Cells (A) MCF7 cells were transfected with the RGC-ΔFos- LacZ p53-dependent reporter construct as well as a control vector or an expression vector for the SirT1-363Y dominant negative mutant. All samples were also transfected with a control plasmid expressing luciferase under the control of the SV promoter. β-galactosidase activity was measured 32 hr after transfection and values were normalized using the luciferase readings. Values correspond to three independent experiments ± SD. (B) H1299 cells (p53 null) were transfected with vectors expressing p53 and mdm2 in the absence or presence of a vector expressing SirT1 (pCMV-SirT1). Cells were treated with increasing concentrations of tenovin-1 for 6 hr, and the levels of p53 and SirT1 were analyzed by western blot using DO1 and antibody 2G1-F7 (Cat. No. 05-707, Upstate), respectively. Note that pCMV-SirT1 encodes SirT1 isoform-1. Endogenous SirT1 isoform-1 was also detected in lanes 1 through 5 upon longer exposure of the blots. The band below ectopic SirT1 could correspond to a SirT1 isoform. (C) MCF-7 cells were treated with 10 μM tenovin-1 for the indicated times and analyzed by western blotting using an antibody against K382-acetylated p53 (Cat. No. 614202, BioLegend) or the DO1 antibody against the N terminus of p53. PCNA was detected as a loading control. (D) H1299 cells transfected with a vector for p53 were treated for 6 hr with the indicated concentrations of tenovin-6. K382-acetylated p53 and total p53 were detected as above. (E) H1299 cells were transfected with a vector for p53 expression (upper panels) or p53R273H (lower panels) in the absence or presence of pCMV-SirT1. Cells were treated for 6 hr with the indicated concentrations of tenovin-1. K382-acetylated p53 and total p53 were detected. (F) H1299 cells were transfected with a vector for wild-type p53 expression (lanes 1, 2, and 3) or p53R273H (lanes 4, 5, and 6). Cells were left untreated (lanes 3 and 4) or treated with 10 μM (lanes 2 and 5) or 20 μM (lanes 1 and 6) tenovin-6 for 6 hr. K382-acetylated p53 and total p53 were detected, and the ratio between the amount of K382-acetylated p53 and the total amount of p53 in each lane was calculated. Note that these ratios do not correspond to the actual fraction of acetylated p53 in cells. Lanes 7, 8, and 9 correspond to loading 1/10 of the amount of protein in samples in lanes 4, 5, and 6, respectively. (G) H1299 cells were transfected with a vector for wild-type p53 expression (lanes 1 through 3) or p53R273H (lanes 4 through 6) in the absence (lanes 1 and 4) or presence (lanes 2, 3, 5, and 6) of ectopic mdm2. In lanes 3 and 6, cells were treated for 6 hr with 10 μM tenovin-1. Total p53 was detected with DO1 antibody. β-gal expression was used as a transfection efficiency and loading control. (H) H1299 cells were treated with 10 μM tenovin-1 for the indicated times. Endogenous p14ARF was detected using a mouse monoclonal antibody (Ab-3 14P03, Neomarkers). PCNA was detected as a loading control.

Article Snippet: Human wild-type p53, p53R273H, and mdm2 expression vectors are described ( ). pCMV-SirT1 vector for SirT1 isoform 1 was obtained from Origene (Cat. No. SC127917).

Techniques: Transfection, Construct, Plasmid Preparation, Expressing, Dominant Negative Mutation, Luciferase, Activity Assay, Western Blot

Journal: Cancer Cell

Article Title: Discovery, In Vivo Activity, and Mechanism of Action of a Small-Molecule p53 Activator

doi: 10.1016/j.ccr.2008.03.004

Figure Lengend Snippet: Tenovin-6 Inhibits the Protein Deacetylase Activities of Purified Sirtuins SirT1 and SirT2 (A–D) Increasing concentrations of tenovin-6 were added to purified human SirT1 (A), SirT2 (B), SirT3 (C), or HDAC8 (D) reaction mixtures. Values correspond to the average enzyme activity of three independent experiments ± SD. Estimated IC 50 values for SirT1, SirT2, and SirT3 in the assay conditions are 21, 10, and 67 μM, respectively. (E) Analysis of tenovin-6's ability to compete for binding sites with the SirT1 FdL acetylated peptide substrate. In vitro SirT1 inhibition assay was carried out with tenovin-6 at 0, 50, and 75 μM with varying FdL concentrations and a constant NAD+ concentration of 1 mM. All assays contained the same amount of DMSO (0.25%). The data is presented as a Lineweaver-Burke plot (where the x axis intercept is −1/K m and the y axis intercept is 1/V max ). Trend lines were then added to create a straight line. The trend lines for 0 μM (triangles), 50 μM (asterisks), and 75 μM (circles) tenovin-6 all have an R 2 values above 0.99. Data points are the average of triplicate experiments. (F) Analysis of tenovin-6's ability to compete for binding sites with SirT1 cosubstrate NAD+. In vitro SirT1 inhibition assay was carried out with tenovin-6 at 0, 25, and 50 μM with varying NAD + concentrations and a constant FdL acetylated peptide concentration of 200 μM. All assays contained the same amount of DMSO (0.25%). The data are presented as a Lineweaver-Burke plot. The trendlines for 0 μM (diamonds), 25 μM (squares), and 50 μM (triangles) tenovin-6 all have an R 2 value above 0.99. Data points are the average of triplicate experiments.

Article Snippet: Human wild-type p53, p53R273H, and mdm2 expression vectors are described ( ). pCMV-SirT1 vector for SirT1 isoform 1 was obtained from Origene (Cat. No. SC127917).

Techniques: Histone Deacetylase Assay, Purification, Activity Assay, Binding Assay, In Vitro, Inhibition, Concentration 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, Expressing, Over Expression, Activity Assay, Western Blot

Journal: Oncotarget

Article Title: SIRT1 promotes metastasis of human osteosarcoma cells

doi: 10.18632/oncotarget.12916

Figure Lengend Snippet: A. Four representative immunohistochemical analyses of SIRT1 expression levels in human osteosarcoma tissues. Four representative cases were subjected to immunohistochemical staining using an anti-SIRT1 antibody, and cryosections were stained with haematoxylin and eosin. B. Representative immunohistochemical analyses of SIRT1 expression levels in human osteosarcoma and adjacent tumour tissues. C. SIRT1 expression levels in 33 detected osteosarcoma tumour and adjacent tissue samples were graded and summarised using pie charts. “-”, negative expression; ‘-/+”, slight expression; “+”, strong expression; and “+”, very strong positive expression.

Article Snippet: The shRNA-expressing lentiviral vector pGFP-V-RS against the SIRT1 gene was obtained from Origene (cat. #: TG309433; Rockville, MD, USA).

Techniques: Immunohistochemical staining, Expressing, Staining

Journal: Oncotarget

Article Title: SIRT1 promotes metastasis of human osteosarcoma cells

doi: 10.18632/oncotarget.12916

Figure Lengend Snippet: A. Death and metastatic rates of 22 patients with different SIRT1 expression grades were analysed. “-”, negative expression; ‘-/+”, slight expression; “+”, strong expression; and “+”, very strong positive expression. B. SIRT1 expression in patients with high- and low-risk osteosarcoma according to the GSE21257 dataset using a SurvExpress analysis. C. Relevance between SIRT1 expression level and patient overall survival using PROGgene V2 analysis.

Article Snippet: The shRNA-expressing lentiviral vector pGFP-V-RS against the SIRT1 gene was obtained from Origene (cat. #: TG309433; Rockville, MD, USA).

Techniques: Expressing

Journal: Oncotarget

Article Title: SIRT1 promotes metastasis of human osteosarcoma cells

doi: 10.18632/oncotarget.12916

Figure Lengend Snippet: A. Western blotting of SIRT1 and β-actin in the MDOS-22, MDOS-19, MDOS-21, MDOS-16, MDOS-26, MDOS-14 and MDOS-27 primary osteosarcoma cell lines. Anti-SIRT1 and anti-β-actin antibodies were used to detect SIRT1 and β-actin expression, respectively. B. Relative SIRT1 protein expression levels in A) were normalised to those of β-actin, as indicated in the histogram. Bars, mean ± standard deviation (SD). C. Transwell migration assay of the MDOS-22, MDOS-19, MDOS-21, MDOS-16, MDOS-26, MDOS-14 and MDOS-27 primary osteosarcoma cell lines. Representative images of migrated cells. D. The number of migrated cells per field in C) was quantified and is shown as a histogram. Bars, mean ± SD.

Article Snippet: The shRNA-expressing lentiviral vector pGFP-V-RS against the SIRT1 gene was obtained from Origene (cat. #: TG309433; Rockville, MD, USA).

Techniques: Western Blot, Expressing, Standard Deviation, Transwell Migration Assay

Journal: Oncotarget

Article Title: SIRT1 promotes metastasis of human osteosarcoma cells

doi: 10.18632/oncotarget.12916

Figure Lengend Snippet: A. Western blotting of SIRT1 expression in KHOS/NP cells after infection with lentivirus- short hairpin RNA (shRNA)-SIRT1 (#1 and #2) or control lentivirus (scramble). B. Transwell migration assay of KHOS/NP cells infected with lentivirus-shRNA-SIRT1 (#1 and #2) or control lentivirus (scramble). Representative images of migrated cells are shown. C. The number of migrated KHOS/NP cells per field was quantified and is shown as a histogram after normalisation. Bars, mean ± standard deviation (SD). D. Western blotting of SIRT1 expression in the MDOS-14 primary osteosarcoma cell line after infection with lentivirus-shRNA-SIRT1 (#1 and #2) or control lentivirus (scramble). E. Transwell migration assay of MDOS-14 cells infected with lentivirus-shRNA-SIRT1 (#1 and #2) or control lentivirus (scramble). Representative images of migrated cells are shown. (C) F. The number of migrated MDOS-14 cells per field was quantified and is shown as a histogram after normalisation. Bars, mean ± SD.

Article Snippet: The shRNA-expressing lentiviral vector pGFP-V-RS against the SIRT1 gene was obtained from Origene (cat. #: TG309433; Rockville, MD, USA).

Techniques: Western Blot, Expressing, Infection, shRNA, Transwell Migration Assay, Standard Deviation

Journal: Oncotarget

Article Title: SIRT1 promotes metastasis of human osteosarcoma cells

doi: 10.18632/oncotarget.12916

Figure Lengend Snippet: A. KHOS/NP cells infected with lentivirus-shRNA-SIRT1 (#2) or control lentivirus (scrambled) were injected into the tail vein of BALB/c (nu/nu) mice (n = 6), and formation of metastatic nodes was determined at week 6. B. Western blotting of SIRT1 expression in KHOS/NP cells after infection with lentivirus-shRNA-SIRT1 (#2) or control lentivirus (scramble). C. Representative haematoxylin and eosin staining of lung tissue is shown. D. The number of metastatic nodes per lung is shown. Bars represent mean ± standard deviation (n = 6). **, p < 0.01.

Article Snippet: The shRNA-expressing lentiviral vector pGFP-V-RS against the SIRT1 gene was obtained from Origene (cat. #: TG309433; Rockville, MD, USA).

Techniques: Infection, shRNA, Injection, Western Blot, Expressing, Staining, Standard Deviation

Journal: Oncotarget

Article Title: SIRT1 promotes metastasis of human osteosarcoma cells

doi: 10.18632/oncotarget.12916

Figure Lengend Snippet: A. Schematic representation comparing the gene expression profiles in KHOS/NP cells. Overlapping smaller circles reflect the 3,275 shared genes (2,662 upregulated and 613 downregulated) induced by lentivirus-shRNA-SIRT1 (#1 and #2). B. Functional annotation clustering of the 3,275 overlapped genes according to their DAVID enrichment score. A higher enrichment score for a group indicates that the gene members in the group are involved in more important terms. C, E. Heatmap display of hierarchical clustering of overlapped genes sorted from Table . A total of 100 genes associated with metastasis whose expression changed ≥ two-fold were clustered. D, F. Real-time polymerase chain reaction analysis was used to validate five upregulated D) and two downregulated F) genes in KHOS/NP cells.

Article Snippet: The shRNA-expressing lentiviral vector pGFP-V-RS against the SIRT1 gene was obtained from Origene (cat. #: TG309433; Rockville, MD, USA).

Techniques: Expressing, shRNA, Functional Assay, Real-time Polymerase Chain Reaction

Journal: Oncotarget

Article Title: SIRT1 promotes metastasis of human osteosarcoma cells

doi: 10.18632/oncotarget.12916

Figure Lengend Snippet: Sirt1 array genes

Article Snippet: The shRNA-expressing lentiviral vector pGFP-V-RS against the SIRT1 gene was obtained from Origene (cat. #: TG309433; Rockville, MD, USA).

Techniques: shRNA

Journal: Oncotarget

Article Title: miR-30a suppresses lung cancer progression by targeting SIRT1

doi: 10.18632/oncotarget.23529

Figure Lengend Snippet: ( A and B ) Western blot analysis of the expression of the SIRT1 protein in 6 pairs of lung cancer (LC) and normal adjacent (LN) tissue samples. A: representative image; B: quantitative analysis. ( C ) Quantitative RT-PCR analysis of the relative expression levels of SIRT1 mRNA in 6 pairs of LC and LN samples. *** P < 0.001.

Article Snippet: A mammalian expression plasmid encoding the human SIRT1 open reading frame (pReceiver-M02- SIRT1) was purchased from GeneCopoeia (Germantown, MD, USA).

Techniques: Western Blot, Expressing, Quantitative RT-PCR

Journal: Oncotarget

Article Title: miR-30a suppresses lung cancer progression by targeting SIRT1

doi: 10.18632/oncotarget.23529

Figure Lengend Snippet: ( A ) Schematic depicting the hypothetical duplexes formed through interactions between the binding sites in the SIRT1 3′-UTR (top) and miR-30a (bottom). The predicted free energy of each hybrid is indicated. The seed recognition sites are denoted, and all nucleotides in these regions are highly conserved across species. ( B ) Quantitative RT-PCR analysis of the miR-30a expression levels in six pairs of LC and LN samples. *** P < 0.001.

Article Snippet: A mammalian expression plasmid encoding the human SIRT1 open reading frame (pReceiver-M02- SIRT1) was purchased from GeneCopoeia (Germantown, MD, USA).

Techniques: Binding Assay, Quantitative RT-PCR, Expressing

Journal: Oncotarget

Article Title: miR-30a suppresses lung cancer progression by targeting SIRT1

doi: 10.18632/oncotarget.23529

Figure Lengend Snippet: ( A ) Quantitative RT-PCR analysis of the expression levels of miR-30a in A549 and H1975 cells transfected with equal doses of the miR-30a mimic (pre-miR-30a), miR-30a inhibitor (anti-miR-30a) or scrambled negative control RNA (pre-miR-control or anti-miR-control). ( B and C ) Western blotting analysis to detect SIRT1 protein levels in A549, and H1975 cells transfected with equal doses of the miR-30a mimic, miR-30a inhibitor or scrambled negative control RNA. (B) representative image; (C) quantitative analysis. ( D ) Quantitative RT-PCR analysis of SIRT1 mRNA levels in A549 and H1975 cells transfected with equal doses of the miR-30a mimic, miR-30a inhibitor or scrambled negative control RNA. ( E and F ) Direct recognition of the SIRT1 3′-UTR by miR-30a. Firefly luciferase reporters containing either wild-type (WT) or mutant (MUT) miR-30a binding sites in the SIRT1 3′-UTR were co-transfected into 293T (E) and A549 (F) cells with equal doses of the miR-30a mimic, miR-30a inhibitor or scrambled negative control RNA. After twenty-four hours post-transfection, the cells were assayed using a luciferase assay kit. Firefly luciferase values were normalized to β-galactosidase activity, and the results were calculated as the ratio of firefly luciferase activity in the miR-30a-transfected cells normalized to the negative control RNA-transfected cells. The results are presented as the mean ± S.E. of three independent experiments. ( * p < 0.05; ** p < 0.01; *** p < 0.005).

Article Snippet: A mammalian expression plasmid encoding the human SIRT1 open reading frame (pReceiver-M02- SIRT1) was purchased from GeneCopoeia (Germantown, MD, USA).

Techniques: Quantitative RT-PCR, Expressing, Transfection, Negative Control, Control, Western Blot, Luciferase, Mutagenesis, Binding Assay, Activity Assay

Journal: Oncotarget

Article Title: miR-30a suppresses lung cancer progression by targeting SIRT1

doi: 10.18632/oncotarget.23529

Figure Lengend Snippet: ( A ) A cell proliferation assay was performed 12, 24, 36 and 48 hours after the transfection of A549 cells with equal doses of the miR-30a mimic or scrambled negative control RNA. ( B ) The cell proliferation assay was performed 12, 24, 36 and 48 hours after the transfection of A549 cells with equal doses of the miR-3a inhibitor or scrambled negative control RNA. ( C ) The cell proliferation assay was performed 12, 24, 36 and 48 hours after the transfection of A549 cells with equal doses of the pre-miR-control plus control plasmid, pre-miR-control plus SIRT1 overexpression plasmid, miR-30a mimic plus control plasmid, or miR-30a mimic plus SIRT1 overexpression plasmid. ( D–F ) The apoptosis assay was performed 24 hours after the transfection of A549 cells with equal doses of the miR-30a mimic, miR-30a inhibitor or scrambled negative control RNA or with equal doses of the pre-miR-control plus control plasmid, pre-miR-control plus SIRT1 overexpression plasmid, miR-30a mimic plus control plasmid, or miR-30a mimic plus SIRT1 overexpression plasmid. (D and E) representative image; (F) quantitative analysis. ( G and H ) Transwell analysis was performed after the transfection of A549 cells with equal doses of the miR-30a mimic, miR-30a inhibitor or scrambled negative control RNA or with equal doses of the pre-miR-control plus control plasmid, pre-miR-control plus SIRT1 overexpression plasmid, miR-30a mimic plus control plasmid, or miR-30a mimic plus SIRT1 overexpression plasmid. G. representative image; H. quantitative analysis. The results are presented as the mean ± S.E. of three independent experience ( * p < 0.05; ** p < 0.01; *** p < 0.005).

Article Snippet: A mammalian expression plasmid encoding the human SIRT1 open reading frame (pReceiver-M02- SIRT1) was purchased from GeneCopoeia (Germantown, MD, USA).

Techniques: Proliferation Assay, Transfection, Negative Control, Control, Plasmid Preparation, Over Expression, Apoptosis Assay

Journal: Oncotarget

Article Title: miR-30a suppresses lung cancer progression by targeting SIRT1

doi: 10.18632/oncotarget.23529

Figure Lengend Snippet: Mice were divided into four groups according to the implanted A549 cells: control cells (CTL), miR-30a overexpressing cells (miR-30a), SIRT1-overexpressing cells (SIRT1), and SIRT1 plus Lenti-miR-30a cells (miR-30a+ SIRT1). ( A ) Representative images of tumors. ( B ) The time course of tumor volume. ( C ) The quantitative analysis of tumor weight. ( D ) Quantitative RT-PCR analysis of the expression levels of miR-30a in tumors from four groups of mice. ( E ) Western blotting analyses of SIRT1 proteins in tumors from four groups of mice. ( F ) HE staining, Ki67 and SIRT1 immunohistochemical staining of tumor tissues in four groups of mice. ( G ) Quantitative analyses of Ki-67-positive and SIRT1-positive signals in the tumor. All data are shown as the means ± S.E. obtained from three separate experiments. ( * p < 0.05; ** p < 0.01; *** p < 0.005).

Article Snippet: A mammalian expression plasmid encoding the human SIRT1 open reading frame (pReceiver-M02- SIRT1) was purchased from GeneCopoeia (Germantown, MD, USA).

Techniques: Control, Quantitative RT-PCR, Expressing, Western Blot, Staining, Immunohistochemical staining

Journal: Oncotarget

Article Title: miR-30a suppresses lung cancer progression by targeting SIRT1

doi: 10.18632/oncotarget.23529

Figure Lengend Snippet: Schematic illustration of the miR-30a-SIRT1 axis

Article Snippet: A mammalian expression plasmid encoding the human SIRT1 open reading frame (pReceiver-M02- SIRT1) was purchased from GeneCopoeia (Germantown, MD, USA).

Techniques: