Journal: Experimental and Therapeutic Medicine

Article Title: Resveratrol‑mediated activation of SIRT1 inhibits the PERK‑eIF2α‑ATF4 pathway and mitigates bupivacaine‑induced neurotoxicity in PC12 cells

doi: 10.3892/etm.2023.12132

Figure Lengend Snippet: RSV protects PC12 cells against BUP-induced cytotoxicity via SIRT1 upregulation. (A) Cell viability in PC12 cells exposed to increasing concentrations of EX527. (B) Morphology of PC12 cells in the control, BUP, RSV + BUP and EX527 + RSV + BUP groups observed under a phase-contrast microscope (magnification, x200; scale bar, 50 µm). (C) Representative western blot images and semi-quantification of SIRT1 protein levels in each group. (D) Cell viability in each group. (E) Representative immunofluorescence images of SIRT1 (green) and cell nuclei (blue) staining (scale bar, 50 µm) and the relative mean intensity of SIRT1 immunofluorescence in each group. Data are presented as the mean ± SEM (n=3). * P<0.05 vs. the control group; # P<0.05 vs. the BUP group; & P<0.05 vs. the RSV + BUP group. RSV, resveratrol; BUP, bupivacaine, SIRT1, sirtuin 1; EX527, SIRT1 inhibitor.

Article Snippet: BUP hydrochloride and RSV were purchased from Sigma-Aldrich (Merck KGaA), while CCT020312 and EX527 were obtained from MedChemExpress.

Techniques: Control, Microscopy, Western Blot, Immunofluorescence, Staining

Journal: Experimental and Therapeutic Medicine

Article Title: Resveratrol‑mediated activation of SIRT1 inhibits the PERK‑eIF2α‑ATF4 pathway and mitigates bupivacaine‑induced neurotoxicity in PC12 cells

doi: 10.3892/etm.2023.12132

Figure Lengend Snippet: RSV protects against BUP-induced apoptosis via the upregulation of SIRT1 expression. (A) Representative western blot images and semi-quantification of Bax, Bcl-2 and cleaved caspase-3 protein levels in the control, BUP, RSV + BUP and EX527 + RSV + BUP groups. Q1 (upper-left quadrant) represents dead cells; Q2 (upper-right quadrant) represents late apoptotic cells; Q3 (lower-right quadrant) represents early apoptotic cells; Q4 (lower-left quadrant) represents live cells. (B) Representative images of the flow cytometric analysis of Annexin V/7-AAD staining and quantification of the apoptotic rates in each group. Data are presented as the mean ± SEM (n=3). * P<0.05 vs. the control group; # P<0.05 vs. the BUP group; & P<0.05 vs. the RSV + BUP group. RSV, resveratrol; BUP, bupivacaine; SIRT1, sirtuin 1; EX527, SIRT1 inhibitor; 7-AAD, 7-aminoactinomycin D.

Article Snippet: BUP hydrochloride and RSV were purchased from Sigma-Aldrich (Merck KGaA), while CCT020312 and EX527 were obtained from MedChemExpress.

Techniques: Expressing, Western Blot, Control, Staining

Journal: Experimental and Therapeutic Medicine

Article Title: Resveratrol‑mediated activation of SIRT1 inhibits the PERK‑eIF2α‑ATF4 pathway and mitigates bupivacaine‑induced neurotoxicity in PC12 cells

doi: 10.3892/etm.2023.12132

Figure Lengend Snippet: RSV inhibits the PERK-eIF2α-ATF4 pathway via the upregulation of SIRT1 expression in BUP-treated PC12 cells. (A) Representative western blot images and semi-quantification of p-PERK/PERK, p-eIF2α/eIF2α and ATF4 protein levels in the control, BUP, RSV + BUP and EX527 + RSV + BUP groups. (B) Representative immunofluorescence images of ATF4 (red) and cell nuclei (blue) staining (scale bar, 50 µm) and relative mean intensity analysis of ATF4 immunofluorescence in each group. Data are presented as the mean ± SEM (n=3). * P<0.05 vs. the control group; # P<0.05 vs. the BUP group; & P<0.05 vs. the RSV+BUP group. RSV, resveratrol; PERK, protein kinase RNA-like ER kinase; eIF2α, eukaryotic translation initiation factor 2 α; ATF4, activating transcription factor 4; SIRT1, sirtuin 1; BUP, bupivacaine; p-, phosphorylated; EX527, SIRT1 inhibitor.

Article Snippet: BUP hydrochloride and RSV were purchased from Sigma-Aldrich (Merck KGaA), while CCT020312 and EX527 were obtained from MedChemExpress.

Techniques: Expressing, Western Blot, Control, Immunofluorescence, Staining

Journal: PLoS ONE

Article Title: Synergistic Interactions between HDAC and Sirtuin Inhibitors in Human Leukemia Cells

doi: 10.1371/journal.pone.0022739

Figure Lengend Snippet: Primary AML cells were incubated with or without sirtuin inhibitors (EX527, sirtinol, cambinol) in the presence or absence of VA at the indicated concentrations. Viability was assessed 48 h later by PI cell staining and flow cytometry. CI values refer to the highest drug concentrations used. CI CT s for each drug combination are presented in the lower insets. An effect of 1 corresponds to 100% specific death whereas an effect of 0 corresponds to 0% specific death.

Article Snippet: EX527 was from Tocris Bioscience (Bristol, UK).

Techniques: Incubation, Staining, Flow Cytometry

Journal: PLoS ONE

Article Title: Synergistic Interactions between HDAC and Sirtuin Inhibitors in Human Leukemia Cells

doi: 10.1371/journal.pone.0022739

Figure Lengend Snippet: A, 3×10 6 primary AML cells/well were plated in 6-well plates and incubated in the presence or absence of 50 µM cambinol, 100 µg/ml VA, or their combination. ΔΨ m was monitored at the indicated time points by TMRE staining and flow cytometry. B, 1×10 6 Jurkat cells were plated in 6-well plates and incubated for 48 h in the presence or absence of 100 µg/ml VA. Thereafter, intracellular Bax content was determined by flow cytometry. C, D, Jurkat cells were transduced with either pMIG or pMIG-Bax. Infected cells were FACS sorted, allowed to expand, and subsequently used for flow cytometric detection of intracellular Bax (C) and for viability assays (D). For these, pMIG- or pMIG-Bax-transduced Jurkat were plated in 96-well plates and incubated in the presence or absence of EX527 or cambinol at the indicated concentrations. Viability was determined by PI staining and flow cytometry 48 h later. A–C, one representative experiment out of three is presented. D, Results are means ± SD of three separate experiments.

Article Snippet: EX527 was from Tocris Bioscience (Bristol, UK).

Techniques: Incubation, Staining, Flow Cytometry, Transduction, Infection

Journal: PLoS ONE

Article Title: Synergistic Interactions between HDAC and Sirtuin Inhibitors in Human Leukemia Cells

doi: 10.1371/journal.pone.0022739

Figure Lengend Snippet: A, B, U937 and 697 cells were transduced to either express an anti-EGFP shRNA (EGFP-sh) or a validated anti-Bax shRNA (Bax-sh). Thereafter cells were used for cell lysate preparation and Bax and γ-tubulin were detected by immunoblotting. B, EGFP-sh or Bax-sh 697 cells were incubated with or without 100 µg/ml VA, 75 µM EX527, 50 µM cambinol, or their combinations. Viability was assessed 36 h later by PI staining and flow cytometry. C, D, EGFP-sh or Bax-sh U937 cells were incubated with or without 100 µg/ml VA, 150 µM EX527, 100 µM cambinol, or their combinations. 36 h later, cells were imaged by light microscopy (D) and cell death was determined by flow cytometric quantification of PI-positive cells (C). A, D, one representative experiment out of three is presented. B, C, Results are means ± SD of three separate experiments. *: p<0.05.

Article Snippet: EX527 was from Tocris Bioscience (Bristol, UK).

Techniques: shRNA, Western Blot, Incubation, Staining, Flow Cytometry, Light Microscopy

Journal: PLoS ONE

Article Title: Cyclocarya paliurus extract activates insulin signaling via Sirtuin1 in C2C12 myotubes and decreases blood glucose level in mice with impaired insulin secretion

doi: 10.1371/journal.pone.0183988

Figure Lengend Snippet: Human recombinant Sirt1 were incubated with CPE (0.5, 2.5, and 5 μg/mL) or resveratrol (5 μM), a Sirt1 activator, as well as substrate peptide, NAD, and developer, and Sirt1 activity was measured (A). C2C12 cells were incubated with CPE (100 μg/mL) with or without treatment with EX527 (40 μM), and 2NBDG uptake (B), Akt phosphorylation at Ser473, and tyrosine phosphorylation of IRS (C) were measured. Sirt1 expression was determined after treatment with Sirt1 siRNA (D). Cells were incubated with CPE (100 μg/mL) with or without Sirt1 knockdown, and 2NBDG uptake (E) and Akt phosphorylation level at Ser473 (F) were determined. The effects of CPE on the phosphorylation level of AMPK (G). Values are expressed as the mean ± SEM ( n = 3–4). * p < 0.05, ** p < 0.01 vs . the vehicle-treated control.

Article Snippet: Cells were pretreated 30 mins with inhibitors, 100 μM HNMPA(AM) 3 (Santa Cruz Biotechnology, Inc.) or 40 μM EX527 (BPS Bioscience, USA) before CPE treatment.

Techniques: Recombinant, Incubation, Activity Assay, Expressing

Journal: Pharmaceutical Biology

Article Title: Nicotinamide mononucleotide alleviates endotoxin-induced acute lung injury by modulating macrophage polarization via the SIRT1/NF-κB pathway

doi: 10.1080/13880209.2023.2292256

Figure Lengend Snippet: NMN activated the SIRT1/NF-κB pathway in sepsis-related ALI. (A–E) Representative bands and quantification of SIRT1, acetylated, phosphorylated NF-κB-p65, and NF-κB-p65. (F–G) Levels of acetylated NF-κB-p65 were shown by immunofluorescence (scale bar = 50 μm). Data are presented as means ± SD and analysed by one-way ANOVA followed with Bonferroni coefficient. ** p < 0.01 compared with control; # p < 0.05, ## p < 0.01 compared with LPS group.

Article Snippet: Mice were pretreated intraperitoneally with NMN (500 mg/kg, Sigma, USA) and/or SIRT1 inhibitor EX-527 (5 mg/kg, Beyotime, China) 1 h before LPS injection as previously described (Caton et al. ; Xu et al. ).

Techniques: Immunofluorescence, Control

Journal: Pharmaceutical Biology

Article Title: Nicotinamide mononucleotide alleviates endotoxin-induced acute lung injury by modulating macrophage polarization via the SIRT1/NF-κB pathway

doi: 10.1080/13880209.2023.2292256

Figure Lengend Snippet: The SIRT1/NF-κB pathway was associated with the NMN-mediated M2 macrophage polarization. (A–G) Representative bands and quantification of SIRT1, acetylated, phosphorylated NF-κB-p65, NF-κB-p65, iNOS, and Arg1. (H–J) The levels of CD86 (for M1 macrophages) and CD206 (for M2 macrophages) were evaluated by flow cytometric analysis. Data are presented as means ± SD and analysed by one-way ANOVA followed with Bonferroni coefficient. * p < 0.05, ** p < 0.01 compared with control; # p < 0.05, ## p < 0.01 compared with LPS group.

Article Snippet: Mice were pretreated intraperitoneally with NMN (500 mg/kg, Sigma, USA) and/or SIRT1 inhibitor EX-527 (5 mg/kg, Beyotime, China) 1 h before LPS injection as previously described (Caton et al. ; Xu et al. ).

Techniques: Control

Journal: bioRxiv

Article Title: NQO1 binds and supports SIRT1 function

doi: 10.1101/139907

Figure Lengend Snippet: Mitochondrial inhibition increases nuclear NQO1 level and its SIRT1 association. A) cytoplasmatic (C) and nuclear (N) NQOldistribution were analyzed in NIH3T3 cells. B) The nuclear fraction of NQO1 was analyzed in NIH3T3 cells following treatment with indicated concentrations of rotenone or antimycin A for 16 hours. C) Subcellular distribution of NQO1 under conditions of mitochondrial inhibition. C2C12 cells were antimycin A treated (10 mM for 16 h) and fixed for immunofluorescence analysis with the NQO1 antibody (Epitomics 2618-1). Nuclear staining is indicated by DAPI. D) H1299 cells in which the endogenous NQO1 gene contains YFP in the second exon (clone 130207p11G9) were antimycin A treated in the presence or absence of EX527 (25 mM), a specific Sirt1 inhibitor. E) HEK293T cells over expressing Flag-SIRT1 and NQO1 were treated with indicated concentrations of rotenone or antimycin A for 16 hours. Following the treatment the nuclear fraction was isolated and the association of NQO1 to SIRT1 in the nucleus was analyzed by flag immunoprecipitation.

Article Snippet: EX527 was from PeproTech.

Techniques: Inhibition, Immunofluorescence, Staining, Expressing, Isolation, Immunoprecipitation