The effect of resveratrol on AMPK activity in C2C12 cells.

<p>(A) Representative immunoblots of phospho-AMPK (Thr172), total AMPK, phospho-acetyl-CoA carboxylase (ACC) (Ser79), total ACC, and GAPDH in C2C12 cells treated with vehicle or 60 µM antimycin A (AA) with or without 30 µM resveratrol (RSV). (B) Quantification of phospho-AMPK (Thr172) level normalized to GAPDH (N = 5). (C) Quantification of phospho-ACC (Ser79) level normalized to GAPDH (N = 5). n.s. = not significant.</p

Effects of p53 knockdown on the functions of SIRT1 modulators.

<p>(A) RT-PCR analysis for p53 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in C2C12 cells transfected with siRNA against negative control (Ctrl) or p53. (B) C2C12 cells transfected with control- (Ctrl-si) or p53-siRNA (p53-si) were treated with vehicle or AA (50 µM) for 24 hours. Apoptotic cells with condensed nuclei were quantified. Data are from three independent experiments. (C and D) The percentage of nuclear-condensed C2C12 cells was analyzed. C2C12 cells transfected with control- (Ctrl-si) or p53-siRNA (p53-si) were pretreated with vehicle or either 60 µM splitomicin (SP in C) or 10 µM RSV in D for 3 hours followed by incubation with 50 µM antimycin A (AA) for 24 hours. Data are from three independent experiments. (E) RT-PCR analysis for p53 mRNA in wild type (p53+/+) or p53-deficient (p53−/−) HCT116 cells. (F) Quantification of apoptotic cells with nuclear condensation in wild-type (p53+/+) or p53-null (p53−/−) HCT116 cells treated with vehicle or AA for 24 hours. Data are from three independent experiments. (G and H) Analysis of apoptosis in HCT116 cells treated with SIRT1 modulators. Wild-type (p53+/+) or p53-deficient (p53−/−) HCT116 cells were pretreated with 60 µM SP (G) or 10 µM RSV (H) for 3 hours and then incubated with 50 µM AA for 24 hours. Data are from three independent experiments. ***p<0.001, **p<0.01, *p<0.05. (I) Representative immunoblots of acetyl-p53 (K379) and p53 in C2C12 cells treated with vehicle or antimycin A (AA, 50 µM) with or without pretreatment with resveratrol (RSV) or Ex527. All cells were treated in the presence of 50 nM of trichostatin A. (J) Representative images of immunostaining for acetyl-p53 (Lys379) in C2C12 cells treated as in A. Scale bar: 20 µm.</p

Resveratrol up-regulates SOD2 and suppresses antimycin A-induced ROS via FOXOs.

<p>(A) C2C12 cells were incubated in the presence or absence of RSV (100 µM) for 6 hours followed by western blotting to analyze the SOD2 protein level. (B) Representative images for SOD2 immunostaining in cultured neonatal rat ventricular myocytes (upper) and quantified data (lower). Cells were incubated in the presence of vehicle (Ctrl), resveratrol (RSV), or nicotinamide (NA) for 48 hours. Scale bar: 10 µm. (C) RT-PCR analyses for FOXO transcription factors in C2C12 cells transfected with negative-control siRNA (Ctrl), or siRNA against FOXO1 (F1), FOXO3a (F3a), or FOXO4 (F4). (D) Quantitative analysis of SOD2 mRNA by real-time RT-PCR in C2C12 cells (N = 6). After being transfected with siRNAs against negative-control (Ctrl), FOXO1 (F1), FOXO3a (F3a), or FOXO4 (F4), the cells were pretreated with resveratrol (RSV, 30 µM) for 3 hours, then incubated with antimycin A (AA, 50 µM) for 24 hours. (E) Immunoblot analysis for SOD2. C2C12 cells transfected with control-siRNA (Ctrl-siRNA) or siRNAs against all three FOXOs (FOXO1, FOXO3a, FOXO4) were preincubated with vehicle or RSV (30 µM) for 3 hours followed by treatment with AA (50 µM) without serum for 24 hours. Total-cell lysates were subjected to immunoblot analysis. (F) Representative images of CM-H₂DCFDA-stained C2C12 cells. Cells transfected with control-siRNA or a mixture of all three FOXO siRNAs (FOXOs-siRNA) were treated with AA (200 µM) for 24 hours after pretreatment with vehicle or RSV (30 µM) for 3 hours. Scale bar: 10 µm. (G) Quantification of CM-H₂DCFDA (DCF) fluorescence. Data are from three independent experiments. (H) Immunoblots for acetyl-FOXO1 and FOXO1 in C2C12 cells. Cells were treated with vehicle, antimycin A (AA, 60 µM), or RSV (30 µM)+AA (left panel), or were incubated with vehicle or nicotinamide (NA, 20 mM) in the presence of trichostatin A (TSA) (right panel). (I) Representative immunoblots for FOXO3a and FOXO4 in C2C12 cells treated with vehicle or AA with or without RSV. ***p<0.001, **p<0.01. n.s. = not significant. a.u. = arbitrary units.</p

Effects of SIRT1 knockdown on functions of SIRT1 modulators.

<p>(A) Immunoblot analysis for SIRT1 in C2C12 cells transfected with control (Ctrl)- or SIRT1-siRNA. (B) Representative images of CM-H₂DCFDA fluorescence. C2C12 cells transfected with control- (Ctrl-) or SIRT1-siRNA were treated with antimycin A (AA) (100 µM) for 3 hours after pretreatment with vehicle or resveratrol (RSV, 10 µM) for 6 hours. Scale bar: 10 µm. (C) Quantification of CM-H₂DCFDA (DCF) fluorescence. (D and E) Quantification of cells with condensed nuclei. C2C12 cells transfected with control- (Ctrl-) or SIRT1-siRNA were treated with AA (200 µM) for 24 hours after pretreatment with vehicle (Ctrl), 10 µM RSV, or 60 µM splitomicin (SP) for 3 hours. Data are from three independent experiments. **p<0.01, *p<0.05. a.u. = arbitrary unit. n.s. = not significant.</p

Effects of SIRT1 modulators on H₂O₂-induced ROS generation and apoptosis.

<p>C2C12 cells were treated with 50 µM H₂O₂ for 24 hours after their pretreatment with vehicle (Ctrl), 5 mM nicotinamide (NA), 60 µM splitomicin (SP), 10 µM resveratrol (RSV), or 1 mM NAD⁺ for 3 hours. (A) Intracellular levels of reactive oxygen species (ROS) were detected by CM-H₂DCFDA (DCF). Data are from three independent experiments. (B and C) Cell death was analyzed by nuclear condensation (B), or by immunostaining for cleaved (active) caspase-3 (C). Data in each panel are from three independent experiments. **p<0.01, *p<0.05. a.u. = arbitrary unit.</p