Fig 1: LKB1-dependent phosphorylation is required for resveratrol-induced Sirt1 activation.A, phosphorylation of Sirt1 in cells treated with resveratrol. HEK293T cells were infected with lentivirus-based particles expressing FLAG-Sirt1 for 12 h, and 48 h later cells were treated with 25 μM resveratrol for 6 h. Cells were collected at indicated time points and FLAG-Sirt1 proteins were immunoprecipitated by anti-FLAG and immunoblotted with anti-phospho-serine/threonine. Immunoprecipitated FLAG-Sirt1 proteins treated with lambda protein phosphatase at 30 °C for 30 min were negative controls. For C2C12 myoblasts, cells were infected with virus particles for 12 h. After infection, cells were continued to culture with fresh medium for 48 h and then were grown in DMEM with 2% horse serum for 4 days to generate C2C12 myotubes. The next steps were same as the mentioned process for HEK293T cells. Representative of three independent experiments. B, in vitro Sirt1 deacetylase activity assay. HEK293T cells were infected with lentivirus-based particles expressing FLAG-Sirt1 for 12 h, and 48 h later cells were treated with 25 μM resveratrol for 6 h. FLAG-Sirt1 proteins were immunoprecipitated by anti-FLAG and eluted by 3×FLAG peptide. Then 100 ng eluted FLAG-Sirt1 was incubated with 1 mM NAD+ and 2 μg GST-tagged K382ac p53 peptide from E. coli at 37 °C for 30 min in 40 μl Sirt1 assay buffer. The acetylation level of K382 site was analyzed by using anti-acetyl-p53 K382 antibody. The precipitated FLAG-Sirt1 pretreated with lambda protein phosphatase and then subjected to in vitro deacetylation assay was the negative control. Representative of three independent experiments. p53 K382 is the deacetylation site of Sirt1 and ack382-p53 is the marker of Sirt1 activity (6, 86). C, phosphorylation of Sirt1 in gene-depleted HEK293T cells treated with resveratrol. HEK293T cells were infected with lentivirus-based particles expressing shRNA control, CAMKKβ shRNA, AMPK shRNA, DYRK1A shRNA, DYRK3 shRNA, JNKs (JNK1 and JNK2) shRNAs, CK2α shRNA, or LKB1 shRNA for 12 h, and 48 h later cells were treated with 25 μM resveratrol for 6 h. Sirt1 proteins were immunoprecipitated by anti-Sirt1 antibody and immunoblotted with anti-phospho-serine/threonine. Representative of three independent experiments. D, phosphorylation of Sirt1 in resveratrol-treated LKB1-depleted HEK293T cells expressing FLAG-tagged WT LKB1. HEK293T cells were infected with lentivirus-based particles expressing shRNA control, AMPK shRNA, or LKB1 shRNA for 12 h, and 36 h later LKB1-depleted cells were infected with virus particles expressing FLAG-LKB1 for 12 h. After 36 h, cells were treated with 25 μM resveratrol for 6 h. Sirt1 proteins were immunoprecipitated by anti-Sirt1 antibody and immunoblotted with anti-phospho-serine/threonine. Representative of three independent experiments. E, the deacetylase activity of Sirt1 in resveratrol-treated LKB1-depleted cells. HEK293T cells were infected with lentivirus-based particles expressing shRNA control, AMPK shRNA, or LKB1 shRNA for 12 h. After 48 h, cells were pretreated with 1 μM doxorubicin for 1 h to increase in vivo K382 acetylation of p53 (deacetylation site of Sirt1) and then were treated with 25 μM resveratrol for 6 h. The whole-cell lysate (WCL) was immunoblotted with anti-acetyl-p53 K382. For C2C12 myoblasts, cells were first were infected with virus particles for 12 h. After infection, cells were continued to culture in fresh DMEM for 48 h and then were grown in DMEM with 2% horse serum for 4 days to generate myotubes. The next steps are same as the mentioned process for HEK293T cells. Representative of three independent experiments. (44). The p53 K382 is the deacetylation site of Sirt1 and ack382-p53 is the marker of Sirt1 activity (6, 86). F, the deacetylase activity of Sirt1 in resveratrol-treated LKB1-depleted HEK293T cells expressing WT LKB1 or kinase-dead mutant. HEK293T cells were infected with lentivirus-based particles expressing shRNA control or LKB1 shRNA for 12 h, and 36 h later LKB1-depleted cells were infected with virus particles expressing FLAG-LKB1 (WT or the KD mutant) for 12 h. After 36 h, cells were treated with 25 μM resveratrol for 6 h. WCL were immunoblotted with anti-acetyl-p53 K382. Endo LKB1, endogenous LKB1; KD, kinase dead; LKB1, Lys78Met; NAD, nicotinamide adenine dinucleotide, Sirt1 cofactor; pT172-AMPK, marker of LKB1 activity; RSV, resveratrol; (44).
Fig 2: LINC00842 reduces deacetylation of PGC-1a by SIRT1 in PDAC cells.a LINC00842 overexpression (LINC-OE) or silence (LINC-KD) did not affect PPARGC1A (PPA) mRNA and protein levels. b LINC00842 expression change altered acetylated PGC-1a (acetyl-PGC-1a) levels. c LINC00842 expression change did not affect SIRT1 mRNA and protein levels. d Reciprocal immunoprecipitation assays show inhibitory effect of LINC00842 on the interaction of PGC-1a with SIRT1. e, f Effect of SIRT1 agonist SRT2104 on the interaction of PGC-1a or its Repression domain (PGC-1a-Rep) with LINC00842. The level of LINC00842 RNA in RNA immunoprecipitation product decreased in a SRT2104 dose-dependent manner. g Results of chromatin isolation by RNA purification (ChIRP) assays using LINC00842-odd, -even or LacZ (negative control) antisense probe sets. SRT2104 treatment reduced the interaction of PGC-1a or its Repression domain with LINC00842. h Immunoprecipitation and immunoblotting assays show inhibitory effect of LINC00842 on PGC-1a deacetylation by SRT2104 (4 µM). i Effects of LINC00842 sense or antisense on in vitro PGC-1a-Lys deacetylation by recombinant human SIRT1 (rhSIRT1) in the presence or absence of NAD+, showing only LINC00842 sense inhibited the deacetylation. Flag was blotted as a loading control. The immunoblots in (g–i) are representative results from 3 independent experiments with similar results. The results shown in (a), (c), (e), and (f) are mean ± SD from 3 independent experiments. The P values were determined by Student’s t-test (two-tailed).
Fig 3: High glucose induces LINC00842 expression via transcription factor YY1 in PDAC cells.a LINC00842 levels in cells cultured with 5 or 25 mM of glucose (n = 3). b LINC00842 levels in cells at different times after switch of glucose dose in culture medium (n = 3). c, d Association of PGC-1a (c, n = 3) or its Repression domain (PGC-1a-Rep, d, n = 3) with LINC00842 determined by RNA immunoprecipitation (RIP) assays in cells cultured with different glucose doses. e Immunoprecipitation and immunoblot analysis of acetyl-PGC-1a levels in cells cultured with 25 mM of glucose at 2 and 4 h and after switch to 5 mM of glucose for 2 and 4 h. f, g Immunoprecipitation and immunoblot analysis of acetyl-PGC-1a levels in cells with LINC00842 overexpression (f) or knockdown (g) exposed to indicated glucose doses for 4 h. h, i Co-immunoprecipitation analysis of PGC-1a and SIRT1 interaction in cells with LINC00842 overexpression (h) or knockdown (i) exposed to indicated glucose doses for 4 h. j Prediction of potential transcription factors in the LINC00842 promoter region. k Relative LINC00842 levels in cells with or without knockdown of each of the 6 transcription factors indicated in (j) (n = 3). l Schema of the putative YY1 binding site in the promoter of LINC00842 gene and the primers used for chromatin immunoprecipitation (ChIP) analysis. The consensus and mutant sequences for YY1 binding were boxed. m ChIP analysis with anti-YY1 antibodies or IgG control. Upper panel shows qPCR results (n = 3) and lower panel are images of agarose gel electrophoresis of the qPCR products. n Luciferase reporter assays in cells co-transfected with the indicated plasmids for 48 h (n = 4). o Luciferase reporter assays in cells cultured in 5 mM of glucose and then switched to 25 mM of glucose for 4 h (n = 4). p Immunoblot analysis of YY1 level in cells exposed to 5 or 25 mM of glucose. q Immunoprecipitation and immunoblot analysis of acetyl-PGC-1a levels in cells transfected with the indicated plasmids or siRNAs and exposed to 25 mM glucose. r Immunoprecipitation and immunoblot analysis of acetyl-PGC-1a levels in cells transfected with the indicated plasmids or antisense oligonucleotide (ASO) of LINC00842 and exposed to 5 mM glucose. The results in (a–d), (k), (m–o) are mean ± SD from at least 3 independent experiments. The P values were determined by Student’s t-test (two-tailed).
Fig 4: Resveratrol promotes the binding affinity of LKB1 and Sirt1.A, in vitro kinase assay using eluted LKB1, STRADα, MO25α, and purified MBP-AMPK protein. FLAG-LKB1 plasmid, FLAG-STRADα plasmid, or FLAG-MO25α plasmid was transfected into HEK293T cells separately. FLAG-tagged proteins were immunoprecipitated from respective lysates with anti-FLAG antibody and eluted by 3×FLAG peptide. We incubated 100 ng FLAG-LKB1, 100 ng FLAG-STRADα, 100 ng FLAG-MO25α, and 2 μg purified MBP-AMPK protein with or without 25 μM resveratrol (RSV) in 40 μl kinase assay buffer at 30 °C for 30 min. Samples were subjected to immunoblotting as indicated. Representative of three independent experiments. B, in vitro kinase assay using eluted LKB1 complex with purified MBP-AMPK protein. FLAG-LKB1 plasmid was transfected into HEK293T cells and cells were treated with 25 μM RSV for 6 h. FLAG-LKB1 proteins were immunoprecipitated by anti-FLAG and were eluted with 3×FLAG peptide. And 100 ng eluted protein and 2 μg purified MBP-AMPK protein were incubated in 40 μl kinase assay buffer at 30 °C for 30 min. Samples were subjected to immunoblotting as indicated. The LKB1 kinase activity was remarked by phosphorylation of Thr172 site of AMPK. The bound fraction about FLAG-LKB1 was analyzed by using anti-STRADα antibody and anti-MO25α antibody. Representative of three independent experiments. C, immunoprecipitation of Sirt1 with LKB1 in RSV-treated C2C12 myotubes. C2C12 myoblast cells were grown in DMEM with 2% horse serum for 4 days. Cells were treated with 25 μM RSV for 6 h. Lysates were subjected to immunoprecipitation using antibody against Sirt1. The precipitates were examined by immunoblotting with anti-LKB1 antibody. D, immunoprecipitation of FLAG-LKB1 with Sirt1 in RSV-treated HEK293T cells. HEK293T cells were infected with lentivirus-based particles expressing FLAG-LKB1 and then were treated with 0 to 100 μM RSV for 6 h. FLAG-LKB1 proteins were immunoprecipitated by anti-FLAG, and the bound fraction was analyzed by using anti-Sirt1 antibody. Representative of three independent experiments. E, immunofluorescent images of subcellular distribution about LKB1 and Sirt1. HA-LKB1 plasmid and FLAG-Sirt1 plasmid were cotransfected into C2C12 cells, and cells were then treated with 25 μM RSV for 30 min. Cells were stained with anti-mouse-HA antibody (red), anti-rabbit-FLAG antibody (green), and DAPI (blue). Scale bar, 5 μm. The scale bar for zoom magnification panels, 0.2 μm. F and G, fluorescence intensity distribution for HA-LKB1 and FLAG-Sirt1 imaging of an arbitrary 200-nm line (as white line indicated) was calculated and plotted. The fluorescence intensity was normalized to the highest value in the region to facilitate the comparison. H, coimmunoprecipitation of LKB1 with Sirt1 in the nuclear or cytoplasmic fractions from treated HEK293T cells with or without RSV. Sirt1 was immunoprecipitated using anti-Sirt1, and the precipitates were analyzed using anti-LKB1. Representative of three independent experiments. I, GST-Sirt1-N (N terminus, aa 1–233), GST-Sirt1-Core (deacetylase core, aa 234–510), and GST-Sirt1-C (C terminus, aa 511–747) proteins were purified from E. coli. Then each of the purified proteins was incubated with recombinant MBP-LKB1, and a pull-down assay for Sirt1 fragments was performed using specific GST antibody. Representative of three independent experiments.
Fig 5: LKB1 activates Sirt1 by direct phosphorylation.A, in vitro kinase assay using recombinant LKB1 kinase and purified GST-Sirt1 truncations. Samples were immunoblotted as indicated. MBP-AMPK incubated with LKB1 kinase was as positive control. Asterisks mark the C terminus of Sirt1. B, LKB1-dependent phosphorylation of Sirt1 is positively correlated with its deacetylase activity. HEK293T cells were infected with lentivirus-based particles expressing FLAG-Sirt1 and FLAG-Sirt1 was immunopurified by anti-FLAG. Precipitated FLAG-Sirt1 (2 µg), 100 ng recombinant LKB1 kinase (WT or kinase dead mutant), and 50 µM ATP were incubated in 40 µl kinase assay buffer at 30 °C for 30 min. FLAG-Sirt1 was spun down and 100 ng FLAG-Sirt1 was incubated with 1 mM NAD+ and 2 µg GST-tagged K382ac p53 peptide at 37 °C for 30 min in 40 µl Sirt1 assay buffer. The acetylation level of K382 site was analyzed by using anti-acetyl-p53 K382 antibody, and the phosphorylation level of Sirt1 was analyzed by using anti-phospho-serine/threonine. Representative of three independent experiments. p53 K382 is the deacetylation site of Sirt1 and ack382-p53 is the marker of Sirt1 activity (6, 86). C, in vitro kinase assay using recombinant LKB1 kinase and purified GST-Sirt1 WT or mutants. Samples were subjected to immunoblotting as indicated. Asterisks mark the proteins of interest. D, HEK293T cells stably expressing Sirt1 shRNA were transfected with lentivirus-based particles expressing FLAG-Sirt1 WT, FLAG-Sirt1 S615A, FLAG-Sirt1 S669A, FLAG-Sirt1 S732A, or FLAG-Sirt1 3A for 12 h. After 48 h, cells were treated with 25 µM resveratrol for 6 h. FLAG-Sirt1 WT or mutant proteins were immunoprecipitated by anti-FLAG. Phosphorylation level of Sirt1 was analyzed by using anti-phospho-serine/threonine. The 3A mutation, Ser615, Ser669, and Ser732 were replaced by Ala. E, phosphorylation of Sirt1 in resveratrol-treated LKB1-depleted cells expressing FLAG-Sirt1 WT or 3A mutant. Cells were infected with lentivirus-based particles expressing Sirt1 shRNA, and particles expressing shRNA control, AMPK shRNA, or LKB1 shRNA for 12 h, and 36 h later gene-depleted cells were infected with virus particles expressing FLAG-Sirt1 WT or 3A mutant for 12 h. After 36 h, cells were treated with 25 µM resveratrol for 6 h. FLAG-Sirt1 proteins were immunoprecipitated and immunoblotted with anti-phospho-serine/threonine. Representative of three independent experiments. The pT172-AMPK, marker of LKB1 activity. F, the activity of Sirt1 WT or mutants that were quantified by using a fluorophore-conjugated acetylated p53 peptide. HEK293T cells were lentivirus-based particles expressing Sirt1 shRNA for 12 h, and 36 h later gene-depleted cells were infected with virus particles expressing FLAG-Sirt1 WT or mutants for 12 h. After 48 h, cells were treated with 25 µM resveratrol for 6 h. FLAG-Sirt1 proteins were immunopurified by anti-FLAG and eluted by 3×FLAG peptide. And 50 ng eluted FLAG-Sirt1 were incubated with 1 mM of NAD+ and 200 µM fluorescently labeled acetylated p53 peptide in Sirt1 assay buffer at 37 °C for 30 min and the reaction was stopped with developer solution containing 2 mM nicotinamide. Sirt1 activity was assessed by measuring the fluorescent emission at 460 nm, following excitation at 360 nm. Data represents mean ± SD. The experiment was repeated six times independently. Statistical significance was determined by Dunnett's multiple comparisons test. ****p (WT, WT RSV) < 0.0001. ***p (WT, 615A) = 0.0002. *p (WT, 669A) = 0.0283. *p (WT, 732A) = 0.0164. ****p (WT, 3A) < 0.0001. 615A, 669A, 732A, and 3A were replaced by Ala. G, LKB1-mediated phosphorylation of Sirt1 is positively correlated with its deacetylase activity in resveratrol-treated Sirt1-depleted cells expressing FLAG-Sirt1 WT or 3A mutant. Cells were infected with lentivirus-based particles expressing Sirt1 shRNA for 12 h, and 36 h later gene-depleted cells were infected with virus particles expressing FLAG-Sirt1 WT or 3A mutant for 12 h. After 36 h, cells were pretreated with 1 µM doxorubicin for 1 h to increase in vivo K382 acetylation of p53 (deacetylation site of Sirt1) and then were treated with 25 µM resveratrol for 6 h. The whole-cell lysate (WCL) was immunoblotted with anti-acetyl-p53 K382. FLAG-Sirt1 proteins were immunoprecipitated and immunoblotted with anti-phospho-serine/threonine. Representative of three independent experiments. The p53 K382 is the deacetylation site of Sirt1 and ack382-p53 is the marker of Sirt1 activity (6, 86). H, in vitro Sirt1 deacetylase activity assay. Baculovirus expressed His-tagged Sirt1 deacetylase (aa 193–747, Abcam, recombinant human Sirt1 protein, ab101130) WT, 3A mutant, or 3D mutant was incubated with 1 mM NAD+ and 2 µg GST-tagged K382ac p53 peptide from Escherichia coli at 37 °C for 30 min in 40 µl Sirt1 assay buffer. Acetylation level of K382 site was analyzed by using anti-acetyl-p53 K382 antibody. Representative of three independent experiments. The 3D mutation, Ser615, Ser669, and Ser732 were replaced by Asp. The p53 K382 is the deacetylation site of Sirt1 and ack382-p53 is the marker of Sirt1 activity (6, 86). 3A, Ser615, Ser669, and Ser732; 615A, Ser615Ala; 669A, Ser669Ala; 732A, Ser732Ala; KD, kinase dead; LKB1, Lys78Met; RSV, resveratrol; Sirt1-C, C terminus, aa 511 to 747; Sirt1-Core, core domain, aa 234 to 510; Sirt1-N, N terminus, aa 1 to 233.
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