Journal: Biology

Article Title: N-acetyl-L-cysteine Prevents Lactate-Mediated PGC1-alpha Expression in C2C12 Myotubes

doi: 10.3390/biology8020044

Figure Lengend Snippet: qPCR analysis of PGC1-alpha and SIRT1 expression in C2C12 myotubes cultured with L-lactate. C2C12 myotubes were cultured for 2 ( a ) and 6 h ( b ) with L-lactate at different concentrations (0, 10, 30, and 50 mM). In the time between lactate culture and sample collection (one hour), cells were cultured with DM. Error bars show standard deviations. * p < 0.05 from 0 mM. All the experiments were performed three independent times with three replicates each time.

Article Snippet: Membranes were then probed at 4 °C overnight in TBS-T containing 0.4% NaN3 and 1:1000 dilutions of specific antibodies against PGC1-alpha (Novus Biologicals, Centennial, CO, USA) and GAPDH (Abcam, Cambridge, UK).

Techniques: Expressing, Cell Culture

Journal: Biology

Article Title: N-acetyl-L-cysteine Prevents Lactate-Mediated PGC1-alpha Expression in C2C12 Myotubes

doi: 10.3390/biology8020044

Figure Lengend Snippet: qPCR analysis of PGC1-alpha, Cyt C, and SIRT1 in C2C12 myotubes cultured with lactate and N-acetyl-L-cysteine (Nac). C2C12 myotubes were cultured for 2 h with L-lactate (0 and 50 mM) in the presence or absence of Nac (2 mM). Samples for gene expression analysis were obtained at 0 ( a ) and 3 h ( b ) after the culture with lactate and Nac. In the time between lactate culture and sample collection (0 to 3 h), cells were cultured with DM. Error bars show standard deviations. * p < 0.05 from 0 mM. All the experiments were performed three independent times with three replicates each time.

Article Snippet: Membranes were then probed at 4 °C overnight in TBS-T containing 0.4% NaN3 and 1:1000 dilutions of specific antibodies against PGC1-alpha (Novus Biologicals, Centennial, CO, USA) and GAPDH (Abcam, Cambridge, UK).

Techniques: Cell Culture, Gene Expression

Journal: Biology

Article Title: N-acetyl-L-cysteine Prevents Lactate-Mediated PGC1-alpha Expression in C2C12 Myotubes

doi: 10.3390/biology8020044

Figure Lengend Snippet: qPCR analysis for intermittent and continuous exposure to lactate in C2C12 myotubes and Western blot of PGC1-alpha after continuous or intermittent exposure to lactate. C2C12 myotubes were cultured with different concentration patterns of L-lactate (see ). Samples for qPCR analysis were obtained 0 ( a ) and 3 h ( b ) after the exposure series. Samples for Western blot ( c ) were obtained 3 h after the exposure series (see ). In the time between lactate culture and sample collection (zero or three hours), cells were cultured with DM. Error bars show standard deviations. * p < 0.05 from non-lactate exposure (0 × 0). RNA experiments were performed three independent times with three replicates each time, and protein experiments with three independent samples.

Article Snippet: Membranes were then probed at 4 °C overnight in TBS-T containing 0.4% NaN3 and 1:1000 dilutions of specific antibodies against PGC1-alpha (Novus Biologicals, Centennial, CO, USA) and GAPDH (Abcam, Cambridge, UK).

Techniques: Western Blot, Cell Culture, Concentration Assay

Journal: Bioscience Reports

Article Title: A conserved mechanism of sirtuin signalling through steroid hormone receptors

doi: 10.1042/BSR20193535

Figure Lengend Snippet: ( A ) A simplified modular structure of human SIRT1 showing locations of the STACs-activation domain (STACs-AD) and the Core Catalytic (deacetylation) Domain (CCD). NTD and CTD are the N-terminal and C-terminal domains respectively. A classical nuclear receptor box (NR-box) is located within the STACs-AD. Position of the partially conserved STACs activation residue E230 is shown for context . The alignment shows putative NR-boxes of SIRT1 (representing orthologues from different mammals) with orthologous sirtuins in the long-lived naked mole rat H. glaber , the short-lived killifish N. furzeri, S. cerevisiae, D. melanogaster and C. elegans . The NR-box core (LxxLL) of SIRT1 is homologous to those of prototypical coregulators GRIP-1 (Glucocorticoid Receptor-Interacting Protein-1), CBP (CREB-Binding Protein), TIF-1 (Transcriptional Intermediary Factor-1) and SRC-1/NCOA1 (Steroid Receptor Coactivator-1/ Nuclear Receptor Coactivator 1). ( B ) SIRT1 NR-box (see text) is a class III ERα-selective interface with a hydrophobic residue (Φ) at -1, and a hydrophilic residue (serine or threonine) at position -2 . In (A) and (B), positions of the aligned sequences within the coregulators are bracketed. ( C ) One-hybrid assay in HEK293 cells to determine activation functions in SIRT1 and its derivatives tethered to the Gal4 DNA-binding domain (Gal4DBD) as shown. Each construct or Gal4DBD (pM backbone) was cotransfected with the minimal promoter vector pFR-Luc (containing 5x Gal4-binding sites, hereafter labelled 5x Gal4-luc) and β-galactosidase (β-gal) as reporter gene and internal control respectively. The transcriptional activities of the indicated constructs were calculated from the activity of Gal4DBD that was set at 100% as reference. 5x Gal4-luc expression levels were normalized with β-gal and plotted in duplicates as means ± S.E/M; they are representative of three separate experiments. Note that wild-type Gal4DBD-STACs-AD Wt and its mutant Gal4DBD-STACs-ADΔNRB are identical except for mutations in the NR-box (LKDLL→ AKDAA), shown with a triangle within this construct. The activity of Gal4DBD-SIRT1 Wt was compared with its deacetylase-defective mutant Gal4DBD-SIRT1 H 363 Y; mutation is shown with a blue dot. Bracketed numbers besides each construct indicate cloned regions (residues) of SIRT1.

Article Snippet: VP16-ER alpha (# 11351) and VP16-ER beta long isoform (#11352), p413TEF-Sir2 (# 51742), 3xERRE/ERE-luciferase (# 37852), SIRT1.1 (# 13735), Flag-SIRT1 (# 1791), Flag-SIRT1 H 363 Y (# 1792) and Gal4-PGC-1 alpha (# 8892) were purchased from Addgene (Cambridge, MA, U.S.A.). pET28-Sir-2.1 was kindly provided by Leonard Guarente (MIT, Cambridge, MA, U.S.A.) while VP16-DAF-12 and its reporter gene lit -1K-TK-luc [ ] were kindly offered by David Mangelsdorf (UT Southwestern Medical Centre, Dallas, TX, U.S.A.).

Techniques: Activation Assay, Binding Assay, Hybrid Assay, Construct, Plasmid Preparation, Activity Assay, Expressing, Mutagenesis, Histone Deacetylase Assay, Clone Assay

Journal: Bioscience Reports

Article Title: A conserved mechanism of sirtuin signalling through steroid hormone receptors

doi: 10.1042/BSR20193535

Figure Lengend Snippet: ( A ) M2H assays were performed in HEK293 cells to determine interactions between VP16-ERα and SIRT1, Sir2 and Sir-2.1 tethered to Gal4DBD. ( B ) SIRT1-PGC-1α transcriptional complexes strongly interact with ERα and ERβ and overide Gal4 promoter repression by SIRT1 alone. M2H assay was repeated with Gal4DBD-SIRT1 and Gal4DBD-PGC-1α, and ERα or ERβ fused to VP16 activation domain. ( C ) M2H assay for interaction between the STACs-AD and VP16-ERα or VP16-ERβ. Gal4DBD-STACs-AD Wt or its NR-box mutant (ΔNRB) were cotransfected with the receptors as indicated. Schematic shows locations of Wt and mutant NR-boxes within the STACs-AD. Statistical significance of differences in interaction (B) or gene expression in response to DMSO or E2 (A–C) are shown with P values: * P ≤ 0.05, ** P ≤ 0.01 and *** P ≤ 0.001; differences are not significant where P values are not shown. 5x Gal4-luc and β-gal activities were determined as described and ER interaction with Gal4DBD was set as reference point. Data were plotted in duplicates as means ± S.E.M and are representative of three independent experiments.

Article Snippet: VP16-ER alpha (# 11351) and VP16-ER beta long isoform (#11352), p413TEF-Sir2 (# 51742), 3xERRE/ERE-luciferase (# 37852), SIRT1.1 (# 13735), Flag-SIRT1 (# 1791), Flag-SIRT1 H 363 Y (# 1792) and Gal4-PGC-1 alpha (# 8892) were purchased from Addgene (Cambridge, MA, U.S.A.). pET28-Sir-2.1 was kindly provided by Leonard Guarente (MIT, Cambridge, MA, U.S.A.) while VP16-DAF-12 and its reporter gene lit -1K-TK-luc [ ] were kindly offered by David Mangelsdorf (UT Southwestern Medical Centre, Dallas, TX, U.S.A.).

Techniques: Activation Assay, Mutagenesis, Expressing

Journal: Bioscience Reports

Article Title: A conserved mechanism of sirtuin signalling through steroid hormone receptors

doi: 10.1042/BSR20193535

Figure Lengend Snippet: Gal4DBD NR-box fusion peptides derived from C. elegans Sir-2.1 ( A,B ), Drosophila dSir2 ( C,D ), fish ( N. furzeri ) Sirt1, Nf Sirt1 ( E,F ), and S. cerevisiae Sir2 ( G,H ) were tested by the M2H assay for interaction with ERα (A, C, E, G) or ERβ (B, D, F, H). HEK293 cells were cotransfected with 5x Gal4-luc and increasing amounts of Gal4DBD NR-box (NRB) plasmids as indicated, or with Gal4DBD as negative control; cells were then treated with DMSO or 100 nM E2. In all cases, read-outs of ER interactions (fold activation) with the Gal4DBD were set as the reference point after normalizing luciferase activity against β-gal internal control. All data were plotted in duplicates as means ± S.E.M; data represent at least three separate experiments. Differences in gene expression between DMSO- and E2-treated cells were statistically significant if P ≤ 0.05.

Article Snippet: VP16-ER alpha (# 11351) and VP16-ER beta long isoform (#11352), p413TEF-Sir2 (# 51742), 3xERRE/ERE-luciferase (# 37852), SIRT1.1 (# 13735), Flag-SIRT1 (# 1791), Flag-SIRT1 H 363 Y (# 1792) and Gal4-PGC-1 alpha (# 8892) were purchased from Addgene (Cambridge, MA, U.S.A.). pET28-Sir-2.1 was kindly provided by Leonard Guarente (MIT, Cambridge, MA, U.S.A.) while VP16-DAF-12 and its reporter gene lit -1K-TK-luc [ ] were kindly offered by David Mangelsdorf (UT Southwestern Medical Centre, Dallas, TX, U.S.A.).

Techniques: Derivative Assay, Negative Control, Activation Assay, Luciferase, Activity Assay, Expressing, IF-P

Journal: Bioscience Reports

Article Title: A conserved mechanism of sirtuin signalling through steroid hormone receptors

doi: 10.1042/BSR20193535

Figure Lengend Snippet: Increasing doses (50, 100 and 200 ng) of Gal4DBD fusion peptides of wild-type (NRBWt) and mutant (NRBMt) SIRT1 NR-boxes were tested in M2H assays in HEK293 cells for interaction with ( A ) VP16-ERα and ( B ) VP16-ERβ; the reporter gene was 5x Gal4-luc. Luciferase expression was normalized to β-gal activity. Graphs were plotted by setting the activity of Gal4DBD interaction with the ERs as reference. P values show the significance of differences in ER interaction, comparing NRBWt with NRBMt; only P values ≤ 0.05 are significant. ( C–F ) Modified M2H assay of SIRT1 NR-box peptide binding specificity by competitive inhibition with wild-type (C,E) and mutant (D,F) NR-box peptides. Interactions were determined through dose-dependent repression of ERα (C,D) and ERβ (E,F) signalling; antagonism was observed with wild-type (C,E) but not with mutant (D,F) NR-box peptides. For panels (C–F), MCF-7 cells were cotransfected with VP16-ERα or VP16-ERβ, the ER reporter gene instead of 5x Gal4-luc, and increasing amounts (50, 100 and 200 ng) of Gal4DBD SIRT1 NR-box peptides. Cells were treated with DMSO or 100 nM E2 and ER reporter gene expression was normalized to β-gal internal control. Fold activation was determined from the activity of Gal4DBD (negative control) set at a value of 1. P values show significant ( P ≤ 0.05) differences between DMSO and E2-treated cells. Data are representative of three independent assays.

Article Snippet: VP16-ER alpha (# 11351) and VP16-ER beta long isoform (#11352), p413TEF-Sir2 (# 51742), 3xERRE/ERE-luciferase (# 37852), SIRT1.1 (# 13735), Flag-SIRT1 (# 1791), Flag-SIRT1 H 363 Y (# 1792) and Gal4-PGC-1 alpha (# 8892) were purchased from Addgene (Cambridge, MA, U.S.A.). pET28-Sir-2.1 was kindly provided by Leonard Guarente (MIT, Cambridge, MA, U.S.A.) while VP16-DAF-12 and its reporter gene lit -1K-TK-luc [ ] were kindly offered by David Mangelsdorf (UT Southwestern Medical Centre, Dallas, TX, U.S.A.).

Techniques: Mutagenesis, Luciferase, Expressing, Activity Assay, Modification, Binding Assay, Inhibition, Activation Assay, Negative Control