Journal: Journal of Biological Chemistry

Article Title: FoxO1 Deacetylation Regulates Thyroid Hormone-induced Transcription of Key Hepatic Gluconeogenic Genes

doi: 10.1074/jbc.m113.504845

Figure Lengend Snippet: FIGURE 3. T3 induced deacetylation of FoxO1 in a SirT1-dependent man- ner.A,T3decreasedacetylationofFoxO1inatime-dependentmanner.TR1- HepG2 cells were cultured with or without T3 (0.1 M) for the indicated time points. Protein was isolated, and Western blotting was performed to observe FoxO1 deacetylation. B, primary mouse hepatocytes were isolated using a standardtwo-stepcollagenaseperfusionmethodandculturedinDMEMcon- taining 10% Dowex-stripped FBS and 1 penicillin/streptomycin with or without T3 (0.1 M) treatment for 16 h. Protein was isolated, and Western blotting was performed to observe FoxO1 deacetylation. C, SirT1 KD signifi- cantly reduced T3-dependent deacetylation of FoxO1. Cells were cultured withcontrolsiRNAorSirT1siRNAfor48hfollowedbyT3treatment(0.1M)for 24 h. Protein was isolated, and Western blotting was done to observe FoxO1 deacetylation. Densitometric values of AcFoxO1 and FoxO1 were normalized with -actin, then the ratio (AcFoxO1/FoxO1) was plotted as relative density (n 3; *, p 0.05). Error bars represent mean S.D.

Article Snippet: B, to overexpress SirT1 in TR 1-HepG2 cells, FLAG-tagged human SirT1 and its deacetylase domain mutant (H363Y), cloned in pECE (Addgene plasmids 1791 and 1792, respectively) were transfected and confirmed at the protein level.

Techniques: Cell Culture, Isolation, Western Blot

Journal: Journal of Biological Chemistry

Article Title: FoxO1 Deacetylation Regulates Thyroid Hormone-induced Transcription of Key Hepatic Gluconeogenic Genes

doi: 10.1074/jbc.m113.504845

Figure Lengend Snippet: FIGURE 4. T3 induced PCK1 and G6PC mRNA expression in a SirT1-depen- dent manner. A, SirT1 was knocked down using three independent siRNAs in TR1-HepG2 cells and confirmed at the protein level. Cells were cultured with controlsiRNAorSirT1siRNAfor48hfollowedbyreplacementofmediumwith T3-depleted medium as indicated under “Experimental Procedures.” Protein was harvested, and SirT1 KD was confirmed by Western blotting. B, to over- expressSirT1inTR1-HepG2cells,FLAG-taggedhumanSirT1anditsdeacety- lase domain mutant (H363Y), cloned in pECE (Addgene plasmids 1791 and 1792, respectively) were transfected and confirmed at the protein level. Cells were treated with empty vector as control or SirT1 vectors for 48 followed by replacement of medium with normal medium as indicated under “Experi- mental Procedures.” Protein was isolated, and SirT1 expression was con- firmed by Western blotting. C, SirT1 deficiency significantly reduced T3-de- pendent increase in PCK1 and G6PC expression. Cells were treated with control siRNA or SirT1 siRNA for 48 h followed by T3 treatment (0.1 M) for 24 h. 48 h after transfection cells were treated with T3 (0.1 M) for 24 h, total mRNAwascollected,andRT-qPCRanalysiswasperformedasindicatedunder “Experimental Procedures.” D, SirT1 overexpression significantly increased T3-induced PCK1 and G6PC expression. After 48 h of transfection cells were treated with T3 (0.1 M) for 24 h, total mRNA was collected, and RT-qPCR analysis was performed as indicated under “Experimental Procedures.” -Ac- tin was used as normalization control (n 3; *, p 0.05). Error bars represent mean S.D.

Article Snippet: B, to overexpress SirT1 in TR 1-HepG2 cells, FLAG-tagged human SirT1 and its deacetylase domain mutant (H363Y), cloned in pECE (Addgene plasmids 1791 and 1792, respectively) were transfected and confirmed at the protein level.

Techniques: Expressing, Cell Culture, Western Blot, Mutagenesis, Clone Assay, Transfection, Plasmid Preparation, Control, Isolation, Over Expression, Quantitative RT-PCR

Journal: Journal of Biological Chemistry

Article Title: FoxO1 Deacetylation Regulates Thyroid Hormone-induced Transcription of Key Hepatic Gluconeogenic Genes

doi: 10.1074/jbc.m113.504845

Figure Lengend Snippet: FIGURE 6. T3 induced FoxO1 recruitment to PCK1 and G6PC promoters in a SirT1-dependent manner. A, ChIP-qPCR analysis revealed that T3 increased FoxO1 recruitment on PCK1 and G6PC promoters at the IRE region (426 to 170 for PCK1 and 237 to 70 for G6PC gene promoter), which is known for FoxO1 binding in relation to gluconeogenesis. SirT1 deficiency significantly reduced T3-dependent binding of FoxO1. Cells were cultured with control siRNA or SirT1 siRNA for 48 h followed by replacement of medium with normal medium as indicated under “Experimental Procedures.” After 72 h of transfection, cells were treated with T3 for 1 h to analyze FoxO1 recruitment. B, ChIP-qPCR analysis of TRE on PCK1 promoter demonstrates that T3 (0.1 M) rapidly increased acetylation of histone H3 and H4 in this region. HDAC activity of SirT1 was also confirmed as evident from increased basal acetylation of histone H3 and H4 during SirT1 KD in TR-HepG2 cells. During SirT1 KD, T3 (0.1 M) further increased acetylation of these histones, which is significant. 2 l of immunoprecipitated DNA (1% input DNA) was used for qPCR analysis as indicated under “Experimental Procedures” (n 3; *, p 0.05). Error bars represent mean S.D.

Article Snippet: B, to overexpress SirT1 in TR 1-HepG2 cells, FLAG-tagged human SirT1 and its deacetylase domain mutant (H363Y), cloned in pECE (Addgene plasmids 1791 and 1792, respectively) were transfected and confirmed at the protein level.

Techniques: ChIP-qPCR, Binding Assay, Cell Culture, Control, Transfection, Activity Assay, Immunoprecipitation

Journal: Journal of Biological Chemistry

Article Title: FoxO1 Deacetylation Regulates Thyroid Hormone-induced Transcription of Key Hepatic Gluconeogenic Genes

doi: 10.1074/jbc.m113.504845

Figure Lengend Snippet: FIGURE 5. T3 increased PCK1, G6PC gene expression, and SirT1-depen- dentdeacetylationofFoxO1inmicewhichalsorequiredTR.A,hypothy- roidism and hyperthyroidism in male C57BL/6 mice (8–10 weeks old) were induced and confirmed as described under “Experimental Procedures.” B, after 14 h of TH injection, liver tissues were harvested, and total mRNA/protein was extracted. RT-qPCR and Western blot analysis were per- formed on EuTH, HypoTH, and HyperTH mice liver tissues as described under “Experimental Procedures.” An increase in PCK1 and G6PC expression in hyperthyroid mouse liver is significantly corroborated by FoxO1 deacetyla- tion.C,Ex527(0.8mg/day/100gofbodyweight,injectedintraperitoneallyfor 3 days) was used to inhibit SirT1 activity in vivo. After the first injection of Ex527, mice were injected subcutaneously with T3 (10 g/kg of body weight/ day for 2 days) along with Ex527. After 3 days, animals were euthanized, and the liver tissues were subjected to Western blot analysis as indicated under “Experimental Procedures.” Ex527-treated hyperthyroid mouse liver tissues showed a significant increase in FoxO1 acetylation. D, inhibition of TH-in- duced FoxO1 deacetylation in TR-null (TR/) mouse liver tissues showed that TR is essential for TH-dependent FoxO1 deacetylation. A description of TR/ mice is as indicated under “Experimental Procedures.” E and F, in vivo FoxO1 KD using hydrodynamic tail vein injection, as indicated under “Exper- imental Procedures,” significantly inhibited induction in PCK1 mRNA in HyperTH mouse livers when compared with EuTH. After TH treatment in FoxO1 KD mice, liver tissues were harvested, and total mRNA/protein was extracted. RT-qPCR and Western blot analysis were performed on EuTH and

Article Snippet: B, to overexpress SirT1 in TR 1-HepG2 cells, FLAG-tagged human SirT1 and its deacetylase domain mutant (H363Y), cloned in pECE (Addgene plasmids 1791 and 1792, respectively) were transfected and confirmed at the protein level.

Techniques: Gene Expression, Injection, Quantitative RT-PCR, Western Blot, Expressing, Activity Assay, In Vivo, Inhibition

Journal: Molecular Oncology

Article Title: JNK‐mediated Ser27 phosphorylation and stabilization of SIRT1 promote growth and progression of colon cancer through deacetylation‐dependent activation of Snail

doi: 10.1002/1878-0261.13143

Figure Lengend Snippet: Overexpression of P‐SIRT1 Ser27 in CRC tissues and its implications for SIRT1 stability and proliferation and migration of colon cancer cells. (A) SW480 cells (5 × 10 6 ) stably expressing mock or SIRT1 vector were injected subcutaneously into the flank of BALB/c nude mice. Photographs show the xenograft tumors resected from mice at the time of harvest (day 50). The tumor volume and the tumor weight of the mice at the time of harvest were measured. Comparison of the means between mock and SIRT1 vector group was determined by Student's t ‐test. The results are shown as the mean ± S.D. of four xenografts for each group: * P < 0.05. Scale bar represents 1 cm. (B) Histological structures in sections from excised xenograft tumors were analyzed by H&E staining (first column). The yellow arrow indicates neovascularization. The expression of SIRT1, IL‐6, and 8 in excised xenograft tumors were examined by immunohistochemical analysis (second to last columns). Each scale bar represents 100 µm. The data are presented as the mean ± S.D. of three independent samples for each group in Fig. S1H: * P < 0.05; ** P < 0.01. (C) Kaplan–Meier survival curve of overall survival in colon cancer patients with high ( n = 497) and low ( n = 506) levels of SIRT1 based on the data collected from the GENT2 database ( http://gent2.appex.kr ). (D) Kaplan–Meier survival curve of disease‐specific survival in colon cancer patients with high ( n = 238) and low ( n = 238) levels of SIRT1 based on the data obtained from the GENT2 database ( http://gent2.appex.kr ) (E) Immunofluorescence staining of tissue microarray containing thirty‐five pairs of colon adenocarcinoma and matched adjacent normal colon tissues was conducted using anti‐SIRT1 and P‐SIRT1 Ser27 antibodies. The adjacent normal area is 1.5 cm distant from the tumor, which was taken by a histologist. Images of H&E‐stained tissue sections were offered by US Biomax Inc. Each scale bar represents 200 µm. (F) The fluorescence intensity was measured by image analysis software ImageJ, and results are shown as a violin plot. Statistics were calculated using both paired t ‐test and Wilcoxon matched‐pairs test. (G) The protein levels of SIRT1 and P‐SIRT1 Ser27 in colon cancer and corresponding normal tissues were measured by Western blot analysis. The data are presented as the mean ± S.D. of eleven pairs of human tissue specimens in Fig. D: * P < 0.05.

Article Snippet: The membranes were then incubated with primary antibodies against SIRT1, Snail, actin (Santa Cruz Biotechnology, Inc.; Dallas, TX, USA); ubiquitin (Life Technologies by Thermo Fisher Scientific Inc.; Waltham, MA, USA); P‐SIRT1 Ser27 , P‐SIRT1 Ser47 , P‐JNK, JNK, acetylated‐lysine, and acetylated‐p53 Lys382 (Cell Signaling Technology; Danvers, MA, USA).

Techniques: Over Expression, Migration, Stable Transfection, Expressing, Plasmid Preparation, Injection, Comparison, Staining, Immunohistochemical staining, Immunofluorescence, Microarray, Fluorescence, Software, Western Blot

Journal: Molecular Oncology

Article Title: JNK‐mediated Ser27 phosphorylation and stabilization of SIRT1 promote growth and progression of colon cancer through deacetylation‐dependent activation of Snail

doi: 10.1002/1878-0261.13143

Figure Lengend Snippet: Comparison of the effects of SIRT1‐WT and SIRT1‐S27A on the tumorigenicity of HCT‐116 cells. (A) HCT‐116 cells (5 × 10 6 ) transfected with mock, SIRT1‐WT, or SIRT1‐S27A vector were inoculated subcutaneously into the flank of BALB/c nude mice. Photographs show the xenograft tumors collected from mice at the end of the experiment (day 20). The tumor volume and the tumor weight of the mice at the time of harvest were measured, and the one‐way ANOVA with Tukey's multiple comparisons test was performed for the statistical comparison. The results are shown as the mean ± S.D. of six xenografts for each group: * P < 0.05; ** P < 0.01. Scale bar represents 1 cm. (B) H&E staining and immunohistochemical analysis of SIRT1, P‐SIRT1 Ser27 , IL‐6, and IL‐8 were performed on sections from resected xenograft tumor. Each scale bar represents 100 µm. For quantification of each target, the DAB intensity was measured by image analysis software FIJI (the enhanced version of imagej2 ). Statistics were calculated using the one‐way ANOVA with Tukey's multiple comparisons test, and results are presented as the mean ± S.D. ( n = 4 per group): ** P < 0.01; *** P < 0.001. (C) Identification of high‐confidence kinase candidates contributing phosphorylation of SIRT1 at serine 27 by gps 5.0 ( http://gps.biocuckoo.cn/ ). (D) Multiple sequence alignment using clustalw software ( https://www.genome.jp/tools‐bin/clustalw ) showing an evolutionarily conserved phosphorylation site of SIRT1 at serine 27 (arrow) between different species. The positions which have a fully conserved residue are indicated by asterisks. (E) HCT‐116 cells were treated with two different concentrations (10 and 20 µ m ) of a JNK inhibitor (SP600125), an ERK inhibitor (U0126) or a p38 inhibitor (SB203580) for 3 h. The protein levels of P‐SIRT1 Ser27 and SIRT1 were measured by Western blot analysis. One‐way ANOVA with Tukey's multiple comparisons test was used to determine the significance. The results are shown as the mean ± S.D. ( n = 3): ** P < 0.01; *** P < 0.001. (F) HCT‐116 cells were treated with CHX (20 μg·mL −1 ) for indicated periods in the absence or presence of SP600125 (20 μ m ). Comparison of the means between two groups was determined by Student's t ‐test. The values are presented as the mean ± S.D. of three independent experiments: *** P < 0.001.

Article Snippet: The membranes were then incubated with primary antibodies against SIRT1, Snail, actin (Santa Cruz Biotechnology, Inc.; Dallas, TX, USA); ubiquitin (Life Technologies by Thermo Fisher Scientific Inc.; Waltham, MA, USA); P‐SIRT1 Ser27 , P‐SIRT1 Ser47 , P‐JNK, JNK, acetylated‐lysine, and acetylated‐p53 Lys382 (Cell Signaling Technology; Danvers, MA, USA).

Techniques: Comparison, Transfection, Plasmid Preparation, Staining, Immunohistochemical staining, Software, Phospho-proteomics, Sequencing, Residue, Western Blot

Journal: Molecular Oncology

Article Title: JNK‐mediated Ser27 phosphorylation and stabilization of SIRT1 promote growth and progression of colon cancer through deacetylation‐dependent activation of Snail

doi: 10.1002/1878-0261.13143

Figure Lengend Snippet: Strong inactivation of Snail in HCT‐116 cells expressing SIRT1‐S27A. (A) Nuclear extracts prepared from HCT‐116 cells transfected with SIRT1‐WT or SIRT1‐S27A were subjected to the transcription factor activation profiling array to analyze the activity of ninety‐six different transcription factors. The top 10 hits are listed in the Table in an ascending order of the SIRT1‐S27A to SIRT1‐WT ratio. The red arrow denotes the top hit whose activity was most reduced in HCT‐116 cells harboring the SIRT1‐S27A mutation. (B) Following transfection of HCT‐116 cells with mock, SIRT1‐WT, or SIRT1‐S27A vector for 48 h, the mRNA (upper panels) and protein (lower panels) levels of Snail were measured by RT‐PCR and immunoblotting analyses, respectively. Differences in the means among the groups were assessed by one‐way ANOVA with Tukey's multiple comparisons test. Data are presented as the mean ± S.D. ( n = 3 per group): ** P < 0.01; *** P < 0.001. NS, not significant. (C) Immunohistochemical (IHC) analysis of Snail was carried out on sections from resected xenograft tumor corresponding to the Fig. . Each scale bar represents 100 µm. The DAB intensity was assessed by image analysis software fiji (the enhanced version of imagej2 ). Statistics were calculated using one‐way ANOVA with Tukey's multiple comparisons test, and the data are presented as the mean ± S.D. ( n = 4 per group): *** P < 0.001. (D) IHC analysis of Snail and P‐SIRT1 Ser27 was performed on sections from resected xenograft tumor corresponding to the Fig. . Each scale bar represents 100 µm. The results are shown as the mean ± S.D. of three independent samples for each group in Fig. B: * P < 0.05; ** P < 0.01.

Article Snippet: The membranes were then incubated with primary antibodies against SIRT1, Snail, actin (Santa Cruz Biotechnology, Inc.; Dallas, TX, USA); ubiquitin (Life Technologies by Thermo Fisher Scientific Inc.; Waltham, MA, USA); P‐SIRT1 Ser27 , P‐SIRT1 Ser47 , P‐JNK, JNK, acetylated‐lysine, and acetylated‐p53 Lys382 (Cell Signaling Technology; Danvers, MA, USA).

Techniques: Expressing, Transfection, Activation Assay, Activity Assay, Mutagenesis, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunohistochemical staining, Software

Journal: Molecular Oncology

Article Title: JNK‐mediated Ser27 phosphorylation and stabilization of SIRT1 promote growth and progression of colon cancer through deacetylation‐dependent activation of Snail

doi: 10.1002/1878-0261.13143

Figure Lengend Snippet: A proposed mechanism underlying contribution of aberrantly stabilized SIRT1 to oncogenicity of human colon cancer cells. JNK‐dependent phosphorylation of SIRT1 at Ser27 contributes to its stabilization and deacetylation of Snail to enhance the production of IL‐6 and IL‐8, thereby promoting proliferation and migration of human colon cancer cells.

Article Snippet: The membranes were then incubated with primary antibodies against SIRT1, Snail, actin (Santa Cruz Biotechnology, Inc.; Dallas, TX, USA); ubiquitin (Life Technologies by Thermo Fisher Scientific Inc.; Waltham, MA, USA); P‐SIRT1 Ser27 , P‐SIRT1 Ser47 , P‐JNK, JNK, acetylated‐lysine, and acetylated‐p53 Lys382 (Cell Signaling Technology; Danvers, MA, USA).

Techniques: Phospho-proteomics, Migration

Journal: International Journal of Molecular Medicine

Article Title: Enhancement in efferocytosis of oxidized low-density lipoprotein-induced apoptotic RAW264.7 cells through Sirt1-mediated autophagy

doi: 10.3892/ijmm.2013.1609

Figure Lengend Snippet: Expression of Sirtuin1 (Sirt1) and autophagy marker proteins in RAW264.7 cells treated with ox-LDL at different concentrations and time points. (A) Representative results of assays of Sirt1, Atg5, Atg7, LC3-I and LC3-II and β-actin abundances in RAW264.7 cells with ox-LDL of designated concentrations (25, 50 and 100 μM) and time points (12, 24 and 48 h) using western blot analysis. (B) Protein expression levels of Sirt1 were analyzed by western blotting by using polyclonal antibodies to Sirt1, Atg5, Atg7, LC3-I and LC3-II, respectively, in order to quantify the expression in RAW264.7 cells. LC3-II/LC3-I ratio was calculated. β-actin was used as an equal loading control. The band value was quantified by densitometric analysis. The expression of all the proteins was increased most at 24 h compared with 12 or 48 h (all P<0.05). Experiments were repeated at least three times. Data are expressed as the mean ± SD in the corresponding bar graph and statistical significance was determined by the Student’s t-test. Columns, mean; error bars, ±SD; # P<0.05, * P<0.01.

Article Snippet: The anti-Sirt1 antibody was obtained from Cell Signaling (Beverly, MA, USA).

Techniques: Expressing, Marker, Western Blot, Control

Journal: International Journal of Molecular Medicine

Article Title: Enhancement in efferocytosis of oxidized low-density lipoprotein-induced apoptotic RAW264.7 cells through Sirt1-mediated autophagy

doi: 10.3892/ijmm.2013.1609

Figure Lengend Snippet: The optimal concentration of ox-LDL for the expression of Sirtuin1 (Sirt1) and autophagy marker proteins in RAW264.7 cells after incubation for 24 h. (A) Representative western blot analysis results of Sirt1, Atg5, Atg7, LC3-I and LC3-II and β-actin abundances in RAW264.7 cells following stimulation with different concentrations of ox-LDL (0, 25, 50, 75 and 100 μM) for 24 h. (B) Protein levels of Sirt1, Atg5, Atg7, LC3-I and LC3-II were analyzed by western blotting in RAW264.7 cells, respectively. The LC3-II/LC3-I ratio was calculated. β-actin was used as an equal loading control. The band value was quantified by densitometric analysis. The expression of all the proteins was increased after incubation with ox-LDL at the designated concentrations compared with 0 μM ox-LDL (all P<0.05). The optimal concentration of ox-LDL was 75 μM compared with the other groups. Experiments were repeated at least three times. Data are expressed as the mean ± SD in the corresponding bar graph and statistical significance was determined by the Student’s t-test. Columns, mean; error bars, ±SD; # P<0.05 vs. 0 μM group, * P<0.01 vs. 0 μM group.

Article Snippet: The anti-Sirt1 antibody was obtained from Cell Signaling (Beverly, MA, USA).

Techniques: Concentration Assay, Expressing, Marker, Incubation, Western Blot, Control

Journal: International Journal of Molecular Medicine

Article Title: Enhancement in efferocytosis of oxidized low-density lipoprotein-induced apoptotic RAW264.7 cells through Sirt1-mediated autophagy

doi: 10.3892/ijmm.2013.1609

Figure Lengend Snippet: Expression of Sirtuin1 (Sirt1) and autophagy marker proteins in ox-LDL-induced apoptotic RAW264.7 cells of different treatments. The apoptotic cells were randomly divided into 6 groups: i) control, ii) 12.5 μM resveratrol (RSV), iii) 50 μM RSV, iv) 5 mM nicotinamide (NAM), v) 20 mM NAM, vi) 3-methyladenine (3-MA) + 12.5 μM RSV. (A) Representative results of assays of Sirt1, Atg5, Atg7, LC3-I, LC3-II and β-actin abundances of 6 groups using western blot analysis. (B) The levels of Sirt1, Atg5, Atg7, LC3-I and LC3-II protein expression were analyzed by western blot analysis by using polyclonal antibodies to Sirt1, Atg5, Atg7, LC3-I and LC3-II to quantify the expression in these groups. LC3-II/LC3-I ratio was calculated. Sirt1 expression was significantly elevated in 12.5 μM RSV group, compared with control group (P=0.002). No statistical significance was observed between the 3-MA + 12.5 μM RSV and control groups following inhibition of autophagy. The levels of all autophagy marker proteins were significantly increased in 12.5 μM RSV group simultaneously, compared with the control group (all P<0.05). β-actin was used as an equal loading control. The band value was quantified by densitometric analysis. Experiments were repeated at least three times. Data are expressed as the mean ± SD in the corresponding bar graph and statistical significance was determined by the Student’s t-test. Columns, mean; error bars, ±SD; # P<0.05 vs. control group, * P<0.01 vs. control group.

Article Snippet: The anti-Sirt1 antibody was obtained from Cell Signaling (Beverly, MA, USA).

Techniques: Expressing, Marker, Control, Western Blot, Inhibition

Journal: International Journal of Molecular Medicine

Article Title: Enhancement in efferocytosis of oxidized low-density lipoprotein-induced apoptotic RAW264.7 cells through Sirt1-mediated autophagy

doi: 10.3892/ijmm.2013.1609

Figure Lengend Snippet: The ox-LDL uptake of apoptotic RAW264.7 cells of different treatments. (A) Representative results of oil red O staining in ox-LDL-induced apoptotic RAW264.7 cells of different treatments. Images were obtained from a fluorescence microscope. Red fluorescence represents the accumulation of ox-LDL in cells. (B) Analysis of ox-LDL uptake in apoptotic RAW264.7 cells of different treatments. Results were shown as fold changes in the proportions of the oil red O-stained positive area compared with the control. The ox-LDL uptake in the 12.5 and 50 μM resveratrol (RSV) group was decreased (P<0.001, P=0.008 vs. control group respectively), and increased in the 20 mM group (P=0.03 vs. control group). The ox-LDL uptake was also decreased in the 3-methyladenine (3-MA) + 12.5 μM RSV group compared with the control group (P<0.001), which was possibly caused by elevation in the level of Sirtuin1 (Sirt1) although the autophagy was inhibited. Experiments were repeated five times. Data are expressed as the mean ± SD in the corresponding bar graph and statistical significance was determined by the Student’s t-test. Columns, mean; error bars, ±SD; # P<0.05 vs. control group, * P<0.01 vs. control group.

Article Snippet: The anti-Sirt1 antibody was obtained from Cell Signaling (Beverly, MA, USA).

Techniques: Staining, Fluorescence, Microscopy, Control

Journal: International Journal of Molecular Medicine

Article Title: Enhancement in efferocytosis of oxidized low-density lipoprotein-induced apoptotic RAW264.7 cells through Sirt1-mediated autophagy

doi: 10.3892/ijmm.2013.1609

Figure Lengend Snippet: Measurement of the efferocytosis of ox-LDL-induced apoptotic RAW264.7 cells of different treatments. (A) Representative results of the efferocytosis of ox-LDL-induced apoptotic cells of different treatments. Images were obtained from a fluorescence microscope. Red fluorescence represents ox-LDL-induced apoptotic RAW264.7 cells using a PI staining. Green CFSE staining fluorescence represents fresh RAW264.7 cells. (B) Analysis of phagocytic index in apoptotic RAW264.7 cells of different treatments. The phagocytic index 12.5 μM resveratrol (RSV) group was markedly increased compared with the control group (P<0.001), indicating that efferocytosis of apoptotic RAW264.7 cells was enhanced by Sirt1-mediated autophagy. The efferocytosis of apoptotic RAW264.7 cells was decreased in the 5 mM nicotinamide (NAM), 20 mM NAM and 3-MA + 12.5 μM RSV groups (all P<0.001 vs. control group). Data are expressed as the mean ± SD in the corresponding bar graph and statistical significance was determined by the Student’s t-test. Experiments were repeated five times. Columns, mean; error bars, ±SD; # P<0.05 vs. control group, * P<0.01 vs. control group.

Article Snippet: The anti-Sirt1 antibody was obtained from Cell Signaling (Beverly, MA, USA).

Techniques: Fluorescence, Microscopy, Staining, Control

Journal: PLoS ONE

Article Title: The dynamic shuttling of SIRT1 between cytoplasm and nuclei in bronchial epithelial cells by single and repeated cigarette smoke exposure

doi: 10.1371/journal.pone.0193921

Figure Lengend Snippet: (A) SIRT1, SIRT6 and HDAC2 protein expressions are shown in cytoplasmic and nuclei fraction. Lamin A/C and α-tubulin was used for loading control of nuclear and cytoplasmic fractions, respectively. (B) SIRT1 localization in BEAS-2B cells was detected by immunocytochemistry. Upper panel showed SIRT1 immunoreactivity, and lower panel indicated negative control (× 40 in BD Pathway 435 bioimager). (C) SIRT1 subcellular localization was examined for the different human primary cells, such as human airway epithelial cells of bronchial origin (hAEC, passage 2 and 4), air-liquid interface cultured human bronchial epithelial cells (ALI) and human pulmonary artery endothelial cells (hPAEC).

Article Snippet: Commercially available reagents were obtained as follows: RPMI medium 1640 (RPMI 1640) (#11875), polymerase chain reaction (PCR) primer for SIRT1 (Hs01009005) and GNB2L1 (Hs00272002) were from Life Technologies (Carlsbad, CA, USA); fetal bovine serum (FBS), complete protease inhibitor cocktail (#11836153001), anti-SIRT6 antibody (#S4197), anti-HDAC2 antibody (#H2663), thiazolyl blue tetrazolium bromide for MTT assay (M2003), L-buthionine-sulfoximine (BSO) (#B2515), AS605240 (#A0233), rapamycin (#R8781), dimethyl biguanide hydrochloride (metformin) (#D150959), NU7026 (N1537), Z-Leu-Leu-Leu-al (MG-132) (#C2211), cycloheximide (CHX) (#C7698), resveratrol (#R5010) and sirtinol (#S7942) were from Sigma-Aldrich Co. LLC (St Louis, MA, USA); anti-SIRT1 antibody (sc-15404), PIK75 (#sc-296089), IC87114 (#sc-364509), anti-Nrf2 antibody (#sc-13032), anti-α-tubulin antibody (#sc-5286), anti-Lamin A/C antibody (#sc-7292) and anti-chromosomal region maintenance 1 (CRM1) antibody (#sc-5595) were from Santa Cruz Biotechnology (Santa Cruz Biotechnology, CA, USA); anti-β-actin antibody (#ab6276) was from Abcam plc. (Cambridge, UK); goat-derived peroxidase-conjugated anti-mouse (#P0447) or anti-rabbit (#P0448) secondary antibodies were from Dako (Cambridge shire, UK); anti-phospho-SIRT1 (Ser47) antibody (#2314), anti-phospho-Akt (Ser47) (p-Akt) antibody (#4060) and anti-total Akt antibody (#4691) were from Cell Signaling Technology (Danvers, MA, USA); GSK2636771 (#S8002), BIRB796 (#S1574) and U1026 (#S1102) were from Selleck biochemicals (Houston, TX, USA); N-acetyl-Leu-Leu-Norleu-al (ALLN) (#208750) and Akt inhibitor (Akt Inhibitor II) were from Millipore (U.K.) Limited (Hertfordshire, U.K.).

Techniques: Control, Immunocytochemistry, Negative Control, Cell Culture

Journal: PLoS ONE

Article Title: The dynamic shuttling of SIRT1 between cytoplasm and nuclei in bronchial epithelial cells by single and repeated cigarette smoke exposure

doi: 10.1371/journal.pone.0193921

Figure Lengend Snippet: (A) After pretreatment with 100 μM BSO for 16 hours, BEAS-2B cells were exposed to the 3% CSE for up to 24 hours. SIRT1 protein and phosphorylated Akt/Akt ratio(p-Akt / Total-Akt) were assayed at different time point by SDS-PAGE / WB in cytoplasmic fraction. (B) SIRT1 protein levels were examined at different concentration of CSE with or without 100 μM BSO pretreatment. All values were mean value ± SEM of at least three experiments. ** p < 0.01, compared with the values of non-treatment group; †† p < 0.01 between the absence or presence of BSO. (C) Various inhibitors against PI3K signaling pathway molecules were added 30 min prior to the CSE exposure (0.1 μM of PIK75 for PI3Kα inhibitor, 10 μM of GSK2636771 for PI3Kβ inhibitor, 10 μM of AS605240 for PI3Kγ inhibitor, 5 μM of IC87114 for PI3Kδ inhibitor, and 0.02 μM of rapamycin for mTOR inhibitor). The Akt activation status (p-Akt / Total-Akt) and SIRT1 protein levels were evaluated by SDS-PAGE / WB. (D, E) The concentration dependent effects of PIK75 on the SIRT1 and Akt phosphorylation levels were examined using CSE exposure with BSO pretreatment model. The ratio of p-AkT/Total-Akt or SIRT1 levels were fitted with the sigmoid-curve, and calculated for the IC 50 of PIK75 (E, respectively). All values were mean value ± SEM of at least three experiments. ** p < 0.01, compared with the values of non-treatment group; †† p < 0.01 between the two groups.

Article Snippet: Commercially available reagents were obtained as follows: RPMI medium 1640 (RPMI 1640) (#11875), polymerase chain reaction (PCR) primer for SIRT1 (Hs01009005) and GNB2L1 (Hs00272002) were from Life Technologies (Carlsbad, CA, USA); fetal bovine serum (FBS), complete protease inhibitor cocktail (#11836153001), anti-SIRT6 antibody (#S4197), anti-HDAC2 antibody (#H2663), thiazolyl blue tetrazolium bromide for MTT assay (M2003), L-buthionine-sulfoximine (BSO) (#B2515), AS605240 (#A0233), rapamycin (#R8781), dimethyl biguanide hydrochloride (metformin) (#D150959), NU7026 (N1537), Z-Leu-Leu-Leu-al (MG-132) (#C2211), cycloheximide (CHX) (#C7698), resveratrol (#R5010) and sirtinol (#S7942) were from Sigma-Aldrich Co. LLC (St Louis, MA, USA); anti-SIRT1 antibody (sc-15404), PIK75 (#sc-296089), IC87114 (#sc-364509), anti-Nrf2 antibody (#sc-13032), anti-α-tubulin antibody (#sc-5286), anti-Lamin A/C antibody (#sc-7292) and anti-chromosomal region maintenance 1 (CRM1) antibody (#sc-5595) were from Santa Cruz Biotechnology (Santa Cruz Biotechnology, CA, USA); anti-β-actin antibody (#ab6276) was from Abcam plc. (Cambridge, UK); goat-derived peroxidase-conjugated anti-mouse (#P0447) or anti-rabbit (#P0448) secondary antibodies were from Dako (Cambridge shire, UK); anti-phospho-SIRT1 (Ser47) antibody (#2314), anti-phospho-Akt (Ser47) (p-Akt) antibody (#4060) and anti-total Akt antibody (#4691) were from Cell Signaling Technology (Danvers, MA, USA); GSK2636771 (#S8002), BIRB796 (#S1574) and U1026 (#S1102) were from Selleck biochemicals (Houston, TX, USA); N-acetyl-Leu-Leu-Norleu-al (ALLN) (#208750) and Akt inhibitor (Akt Inhibitor II) were from Millipore (U.K.) Limited (Hertfordshire, U.K.).

Techniques: SDS Page, Concentration Assay, Activation Assay, Phospho-proteomics

Journal: PLoS ONE

Article Title: The dynamic shuttling of SIRT1 between cytoplasm and nuclei in bronchial epithelial cells by single and repeated cigarette smoke exposure

doi: 10.1371/journal.pone.0193921

Figure Lengend Snippet: BEAS-2B cells were pretreated with or without 100 μM BSO in the presence or absence of 3% CSE. After 24 hours, localization of SIRT1, SIRT6, HDAC2 and CRM1 in cytoplasm (A) and nuclei (B) were examined by SDS-PAGE / WB. (C) SIRT1 localization after CSE exposure for 24 hours in the presence of BSO in BEAS-2B cells were detected by immunocytochemistry (× 40 in BD Pathway 435 bioimager). (D-G) In the 3% CSE exposure with 100 μM BSO pretreatment model, the SIRT1 shuttling was examined in the human primary bronchial epithelial cells (D, E) or air-liquid interface cultured human bronchial epithelial cells (F, G). (H, I) After CSE exposure with BSO pretreatment in BEAS2B, the effect of PIK75 (0.1 μM) on the SIRT1 shuttling was examined. (J) SIRT1 in nuclear fraction after nicotine (10 μM) or anisomycin (10 μg / ml) treatment for 6 hours in BEAS-2B. (K) Effects of Akt inhibitor (10 μM) on SIRT1 protein in nuclei from cells stimulated with 3% CSE for 24 hours. (L) Effects of N-acetyl cysteine (NAC, 10 mM) and SP-600125 (SP, 10 μM) on SIRT1 protein in nuclei from cells stimulated with 3% CSE for 24 hours. All values were mean value ± SEM of at least three experiments. * p < 0.05, ** p < 0.01, compared with the values of non-treatment group; †† p < 0.01 between the two groups.

Article Snippet: Commercially available reagents were obtained as follows: RPMI medium 1640 (RPMI 1640) (#11875), polymerase chain reaction (PCR) primer for SIRT1 (Hs01009005) and GNB2L1 (Hs00272002) were from Life Technologies (Carlsbad, CA, USA); fetal bovine serum (FBS), complete protease inhibitor cocktail (#11836153001), anti-SIRT6 antibody (#S4197), anti-HDAC2 antibody (#H2663), thiazolyl blue tetrazolium bromide for MTT assay (M2003), L-buthionine-sulfoximine (BSO) (#B2515), AS605240 (#A0233), rapamycin (#R8781), dimethyl biguanide hydrochloride (metformin) (#D150959), NU7026 (N1537), Z-Leu-Leu-Leu-al (MG-132) (#C2211), cycloheximide (CHX) (#C7698), resveratrol (#R5010) and sirtinol (#S7942) were from Sigma-Aldrich Co. LLC (St Louis, MA, USA); anti-SIRT1 antibody (sc-15404), PIK75 (#sc-296089), IC87114 (#sc-364509), anti-Nrf2 antibody (#sc-13032), anti-α-tubulin antibody (#sc-5286), anti-Lamin A/C antibody (#sc-7292) and anti-chromosomal region maintenance 1 (CRM1) antibody (#sc-5595) were from Santa Cruz Biotechnology (Santa Cruz Biotechnology, CA, USA); anti-β-actin antibody (#ab6276) was from Abcam plc. (Cambridge, UK); goat-derived peroxidase-conjugated anti-mouse (#P0447) or anti-rabbit (#P0448) secondary antibodies were from Dako (Cambridge shire, UK); anti-phospho-SIRT1 (Ser47) antibody (#2314), anti-phospho-Akt (Ser47) (p-Akt) antibody (#4060) and anti-total Akt antibody (#4691) were from Cell Signaling Technology (Danvers, MA, USA); GSK2636771 (#S8002), BIRB796 (#S1574) and U1026 (#S1102) were from Selleck biochemicals (Houston, TX, USA); N-acetyl-Leu-Leu-Norleu-al (ALLN) (#208750) and Akt inhibitor (Akt Inhibitor II) were from Millipore (U.K.) Limited (Hertfordshire, U.K.).

Techniques: SDS Page, Immunocytochemistry, Cell Culture

Journal: PLoS ONE

Article Title: The dynamic shuttling of SIRT1 between cytoplasm and nuclei in bronchial epithelial cells by single and repeated cigarette smoke exposure

doi: 10.1371/journal.pone.0193921

Figure Lengend Snippet: (A) The messenger RNA levels of superoxide dismutase 2 (SOD2), SOD3, NADPH quinone oxidoreductase-1 (NQO-1) and hemoxygenase-1 (HO-1), all of which were normalized to mRNA expression of a GNB2L1 housekeeping gene, were assessed by the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) 24 hours after 3% CSE exposure with BSO. All values were mean values ± SEM of at least three experiments. * p < 0.05, ** p < 0.01, compared with the values of non-treatment group; †† p < 0.01, compared between the two groups. (B) The protein levels of HO-1, NQO-1 and SOD2 were assessed by SDS-PAGE/WB. (C) SIRT1 and FOXO3a protein in cytoplasmic fraction (top) and nuclear fraction (bottom) 24 hours after 3% CSE exposure with BSO. PIK75 (0.1 μM) was also treated before CSE exposure. The band density of SIRT1 and FOXO3a were also calculated and corrected to that of Lamin A/C. * p < 0.05, ** p < 0.01, compared with the values of BSO only group; † p < 0.05, compared between with or without PIK75.

Article Snippet: Commercially available reagents were obtained as follows: RPMI medium 1640 (RPMI 1640) (#11875), polymerase chain reaction (PCR) primer for SIRT1 (Hs01009005) and GNB2L1 (Hs00272002) were from Life Technologies (Carlsbad, CA, USA); fetal bovine serum (FBS), complete protease inhibitor cocktail (#11836153001), anti-SIRT6 antibody (#S4197), anti-HDAC2 antibody (#H2663), thiazolyl blue tetrazolium bromide for MTT assay (M2003), L-buthionine-sulfoximine (BSO) (#B2515), AS605240 (#A0233), rapamycin (#R8781), dimethyl biguanide hydrochloride (metformin) (#D150959), NU7026 (N1537), Z-Leu-Leu-Leu-al (MG-132) (#C2211), cycloheximide (CHX) (#C7698), resveratrol (#R5010) and sirtinol (#S7942) were from Sigma-Aldrich Co. LLC (St Louis, MA, USA); anti-SIRT1 antibody (sc-15404), PIK75 (#sc-296089), IC87114 (#sc-364509), anti-Nrf2 antibody (#sc-13032), anti-α-tubulin antibody (#sc-5286), anti-Lamin A/C antibody (#sc-7292) and anti-chromosomal region maintenance 1 (CRM1) antibody (#sc-5595) were from Santa Cruz Biotechnology (Santa Cruz Biotechnology, CA, USA); anti-β-actin antibody (#ab6276) was from Abcam plc. (Cambridge, UK); goat-derived peroxidase-conjugated anti-mouse (#P0447) or anti-rabbit (#P0448) secondary antibodies were from Dako (Cambridge shire, UK); anti-phospho-SIRT1 (Ser47) antibody (#2314), anti-phospho-Akt (Ser47) (p-Akt) antibody (#4060) and anti-total Akt antibody (#4691) were from Cell Signaling Technology (Danvers, MA, USA); GSK2636771 (#S8002), BIRB796 (#S1574) and U1026 (#S1102) were from Selleck biochemicals (Houston, TX, USA); N-acetyl-Leu-Leu-Norleu-al (ALLN) (#208750) and Akt inhibitor (Akt Inhibitor II) were from Millipore (U.K.) Limited (Hertfordshire, U.K.).

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, SDS Page

Journal: PLoS ONE

Article Title: The dynamic shuttling of SIRT1 between cytoplasm and nuclei in bronchial epithelial cells by single and repeated cigarette smoke exposure

doi: 10.1371/journal.pone.0193921

Figure Lengend Snippet: (A) In CSE exposure with BSO pretreatment model, the effects of different concentration of recurrent CSE in the presence or absence of repeated 0.1 μM MG-132 were examined. (B) The effect of 0.3% CSE in the presence of 0.1 μM MG-132 on SIRT1 shuttling under the oxidative stress was confirmed. (C) Cellular oxidative stress determined as malondialdehyde in 3% CSE stimulated and in repeated CSE priming with 3% CSE stimulated cells. (D) SIRT1 proteins in nuclei [N] and cytoplasm [C] in CSE exposed primary bronchial epithelial cells from subjects with or without COPD. (E) Nuclear to cytoplasmic (N/C) ratio of SIRT1 in experiments (D). The data were shown as the relative ratio of that of non-treatment group. * p < 0.05, ** p < 0.01, compared with the value of non-treatment group; † p < 0.05, †† p < 0.01, compared between the two group.

Article Snippet: Commercially available reagents were obtained as follows: RPMI medium 1640 (RPMI 1640) (#11875), polymerase chain reaction (PCR) primer for SIRT1 (Hs01009005) and GNB2L1 (Hs00272002) were from Life Technologies (Carlsbad, CA, USA); fetal bovine serum (FBS), complete protease inhibitor cocktail (#11836153001), anti-SIRT6 antibody (#S4197), anti-HDAC2 antibody (#H2663), thiazolyl blue tetrazolium bromide for MTT assay (M2003), L-buthionine-sulfoximine (BSO) (#B2515), AS605240 (#A0233), rapamycin (#R8781), dimethyl biguanide hydrochloride (metformin) (#D150959), NU7026 (N1537), Z-Leu-Leu-Leu-al (MG-132) (#C2211), cycloheximide (CHX) (#C7698), resveratrol (#R5010) and sirtinol (#S7942) were from Sigma-Aldrich Co. LLC (St Louis, MA, USA); anti-SIRT1 antibody (sc-15404), PIK75 (#sc-296089), IC87114 (#sc-364509), anti-Nrf2 antibody (#sc-13032), anti-α-tubulin antibody (#sc-5286), anti-Lamin A/C antibody (#sc-7292) and anti-chromosomal region maintenance 1 (CRM1) antibody (#sc-5595) were from Santa Cruz Biotechnology (Santa Cruz Biotechnology, CA, USA); anti-β-actin antibody (#ab6276) was from Abcam plc. (Cambridge, UK); goat-derived peroxidase-conjugated anti-mouse (#P0447) or anti-rabbit (#P0448) secondary antibodies were from Dako (Cambridge shire, UK); anti-phospho-SIRT1 (Ser47) antibody (#2314), anti-phospho-Akt (Ser47) (p-Akt) antibody (#4060) and anti-total Akt antibody (#4691) were from Cell Signaling Technology (Danvers, MA, USA); GSK2636771 (#S8002), BIRB796 (#S1574) and U1026 (#S1102) were from Selleck biochemicals (Houston, TX, USA); N-acetyl-Leu-Leu-Norleu-al (ALLN) (#208750) and Akt inhibitor (Akt Inhibitor II) were from Millipore (U.K.) Limited (Hertfordshire, U.K.).

Techniques: Concentration Assay

Journal: PLoS ONE

Article Title: The dynamic shuttling of SIRT1 between cytoplasm and nuclei in bronchial epithelial cells by single and repeated cigarette smoke exposure

doi: 10.1371/journal.pone.0193921

Figure Lengend Snippet: (A) In the 3% CSE exposure with 100 μM BSO pretreatment model, the effect of the SIRT1 inhibitor sirtinol (10 μg/ml) on SIRT1 shuttling and on the mRNA levels of SOD2 (B, left) and SOD3 (B, right) were examined. All values are mean values ± SEM of at least three experiments. * p < 0.05, ** p < 0.01, compared with the values of non-treatment group; † p < 0.05, †† p < 0.01, compared between two groups. (C) The various kinase inhibitors were added 30 min prior to the final 3% CSE exposure (0.1 μM of PIK75 [P] for PI3Kα, 10 μM of GSK2636771 [G] for PI3Kβ, 10 μM of AS605240 [A] for PI3Kγ, 5 μM of IC87114 [I] for PI3Kδ, 0.02 μM of rapamycin [R] for mTOR, 0.1μM of BIRB786 [B] for p38MAPK and 0.1μM of U0126 [U] for ERK signaling. SIRT1 and FOXO3a protein levels in nuclei were evaluated by SDS-PAGE / WB.

Article Snippet: Commercially available reagents were obtained as follows: RPMI medium 1640 (RPMI 1640) (#11875), polymerase chain reaction (PCR) primer for SIRT1 (Hs01009005) and GNB2L1 (Hs00272002) were from Life Technologies (Carlsbad, CA, USA); fetal bovine serum (FBS), complete protease inhibitor cocktail (#11836153001), anti-SIRT6 antibody (#S4197), anti-HDAC2 antibody (#H2663), thiazolyl blue tetrazolium bromide for MTT assay (M2003), L-buthionine-sulfoximine (BSO) (#B2515), AS605240 (#A0233), rapamycin (#R8781), dimethyl biguanide hydrochloride (metformin) (#D150959), NU7026 (N1537), Z-Leu-Leu-Leu-al (MG-132) (#C2211), cycloheximide (CHX) (#C7698), resveratrol (#R5010) and sirtinol (#S7942) were from Sigma-Aldrich Co. LLC (St Louis, MA, USA); anti-SIRT1 antibody (sc-15404), PIK75 (#sc-296089), IC87114 (#sc-364509), anti-Nrf2 antibody (#sc-13032), anti-α-tubulin antibody (#sc-5286), anti-Lamin A/C antibody (#sc-7292) and anti-chromosomal region maintenance 1 (CRM1) antibody (#sc-5595) were from Santa Cruz Biotechnology (Santa Cruz Biotechnology, CA, USA); anti-β-actin antibody (#ab6276) was from Abcam plc. (Cambridge, UK); goat-derived peroxidase-conjugated anti-mouse (#P0447) or anti-rabbit (#P0448) secondary antibodies were from Dako (Cambridge shire, UK); anti-phospho-SIRT1 (Ser47) antibody (#2314), anti-phospho-Akt (Ser47) (p-Akt) antibody (#4060) and anti-total Akt antibody (#4691) were from Cell Signaling Technology (Danvers, MA, USA); GSK2636771 (#S8002), BIRB796 (#S1574) and U1026 (#S1102) were from Selleck biochemicals (Houston, TX, USA); N-acetyl-Leu-Leu-Norleu-al (ALLN) (#208750) and Akt inhibitor (Akt Inhibitor II) were from Millipore (U.K.) Limited (Hertfordshire, U.K.).

Techniques: SDS Page