1. Inhibitory effect of polyphenol cyanidin on TNF-α-induced apoptosis through multiple signaling pathways in endothelial cells 
Jin-Wen Xu, Katsumi Ikeda, Yukio Yamori 
Atherosclerosis 
Volume 193, Issue 2, Pages (August 2007)
DOI: /j.atherosclerosis
Copyright © 2006 Elsevier Ireland Ltd Terms and Conditions

2. Fig. 1
Cyanidin blocks the TNF-α-induced apoptosis in BAECs. (A) Exposure of the serum-starved BAECs (104cells/well) to TNF-α at 50ng/mL for 24h and co-treatment with or without the indicated concentration of cyanidin. Dead cell number was assessed by Trypan Blue staining assay. Data are the mean±S.E.M. (n=6, *p<0.01). (B) TUNEL staining was performed using an in situ cell death detection kit according to the manufacturer's instructions. Arrows indicate the positive cells dyed by TUNEL staining. (C) BAECs were challenged with TNF-α at 50ng/mL and co-treated with or without the indicated concentration of cyanidin for 24h. The cleaved caspase-3 and cleaved poly(ADP-ribose)polymerase (RARP) were assayed by Western blotting using anti-caspase-3 or anti-RARP antibodies. (Top) an original blot; (bottom) results of densitometric analyses. Data are the mean±S.E.M. (n=3).
Atherosclerosis  , DOI: ( /j.atherosclerosis )
Copyright © 2006 Elsevier Ireland Ltd Terms and Conditions

3. Fig. 2
Inhibition of Akt, ERK1/2, Src kinases repressed amelioration of TNF-α-induced apoptosis by cyanidin. (A) BAECs were pretreatment with or without catalase at 10,000U/mL or N-acetylcysteine (NAC) at 500μmol/L and then stimulated with cyanidin at 5μmol/L for 10min. (B) BAECs were incubated with or without pp2 at 20μmol/L, wortmannin at 1μmol/L, or PD98059 at 20μmol/L for 30min and exposed to TNF-α at 50ng/mL and co-treated with or without 50μmol/L of cyanidin for 24h. (C) BAECs were transfected with dominant negative Akt cDNA, then challenged with TNF-α and co-treated with or without cyanidin for 24h. Transfections are described in detail under Section 2. (Top) an original blot; (bottom) results of densitometric analyses. Data are the mean±S.E.M (each n=3, *p<0.01; #p<0.01).
Atherosclerosis  , DOI: ( /j.atherosclerosis )
Copyright © 2006 Elsevier Ireland Ltd Terms and Conditions

4. Fig. 3
NO donor and cyanidin increases expression of thioredoxin (Trx). (A) Mouse kidney endothelial cells were treated with the NO donor SNAP at 50μmol/L for 4 and 8h. (B) After transformation with siRNA of PKG and of control, mouse kidney endothelial cells were treated with or without the NO donor SNAP at 50μmol/L for 4h. (C) BAECs were incubated with cyanidin at 1μmol/L for periods indicated. (D) BAECs were incubated with cyanidin at dose indicated for 4h. (E) BAECs were pretreated with or without 200μmol/L of KT5823 for 30min and stimulated with 5μmol/L of cyanidin. (F) BAECs were exposed to TNF-α at 50ng/mL and co-incunated with or without 50μmol/L of cyanidin for 24h. The Trx expression and S-nitrosylation were assayed by Western blotting using anti-Trx or anti-S-nitrosocysteine antibodies. Data from densitometric analyses are the mean±S.E.M. (each n=3, *p<0.01).
Atherosclerosis  , DOI: ( /j.atherosclerosis )
Copyright © 2006 Elsevier Ireland Ltd Terms and Conditions

5. Fig. 4
Antioxidant effect of cyanidin via the NO-cyclic GMP-dependent protein kinase pathway. (A) BAECs (106cells/well) were incubated with or without 200μmol/L of KT5823 and exposed to TNF-α at 50ng/mL and co-treated with or without 50μmol/L of cyanidin for 24h. The intracellular glutathione was determined using a gluthathione assay kit. Data are the mean±S.E.M. (each n=3, *p<0.01). (B) 4-hydroxynonenal (4-HNE), a major aldehydic product of lipid peroxidation in the samples from the Fig. 4C were assayed by Western blotting using anti-4-HNE antibody.
Atherosclerosis  , DOI: ( /j.atherosclerosis )
Copyright © 2006 Elsevier Ireland Ltd Terms and Conditions

6. Fig. 5
Role of S-nitrosylation in cyanidin inhibition of the TNFα-induced apoptosis pathway. (A) BAECs were challenged with TNF-α at 50ng/mL and co-treated with or without 50μmol/L of cyanidin for 24h. The eNOS expression, S-nitrosylated caspase-3 and acetylated p53 were assayed by Western blotting using anti-eNOS, anti-S-nitrosocysteine or anti-acetylated p53 antibodies. (B) BAECs were incubated with or without 200μmol/L of KT5823, an inhibitor of cyclic GMP-dependent protein kinase, or 10μmol/L of L-NAME, an inhibitor of eNOS, exposed to TNF-α at 50ng/mL and co-treated with or without 50μmol/L of cyanidin for 24h. The p53 expression, cleaved caspase-3 and S-nitrosylated caspase-3 were assayed by Western blotting using anti-p53, anti-S-nitrosocysteine or anti-caspase-3 antibodies. Data from densitometric analyses are the mean±S.E.M. (each n=3, *p<0.01; #p<0.01).
Atherosclerosis  , DOI: ( /j.atherosclerosis )
Copyright © 2006 Elsevier Ireland Ltd Terms and Conditions

7. Fig. 6
Possible scenario of the signaling events in inhibition of cyanidin on the TNF-α-induced apoptosis.
Atherosclerosis  , DOI: ( /j.atherosclerosis )
Copyright © 2006 Elsevier Ireland Ltd Terms and Conditions