P ikb

Total protein was extracted and quantified from heart tissue using the radioimmunoprecipitation assay (RIPA) buffer containing 0.5% protease and phosphatase inhibitor cocktails (Sigma‐Aldrich, #P8340 #P0044). Proteins were denatured and separated on polyacrylamide gels (BioRad, #4561093EDU) using SDS‐PAGE before transfer on PVDF membranes (ThermoScientific, Waltham, MA, #PI88585) as previously described (Ojo et al., 2021 (link)). Membranes were incubated overnight at 4°C in primary antibodies: (p‐eNOS Ser1119 [ThermoFisher, #PIPA564613]; NOS3 [ThermoFisher, #4904]; p‐IKB [Santa Cruz Biotechnology, Dallas, TX, #SC‐8404]; PI3K [Santa Cruz Biotechnology, #SC‐365290]; IKB [Santa Cruz Biotechnology, #SC‐1643]; p‐NFkBp65 Ser536 [#sc‐136548, Santa Cruz Biotechnology]; NFkBp65 [Santa Cruz Biotechnology, #SC‐8008]; and GAPDH [#60004–1, Proteintech, Rosemont IL]) diluted in 5% bovine serum albumin (BSA). Membranes were washed in PBS and incubated for 1 h in anti‐rabbit (Cell Signaling Technology, Danvers, MA #7074) or anti‐mouse (Cell Signaling Technology, #7076) IgG HRP‐linked antibody diluted in 5% milk solution. Proteins were detected using the SuperSignal West Femto Maximum Sensitivity Substrate (ThermoScientific, #34095). Band signals were captured with the ChemiDoc imaging system (BioRad) and quantified with the Image J software, v 1.8.0.

Avenanthramide C was purchased from Sigma–Aldrich (St. Louis, MO, United States; cat. no. 36465) and dissolved in dimethyl sulfoxide (DMSO) (St. Louis, MO, United States; cat.no. D2650) used in doses of 1 μM. It has a purity of 98% by SDS-PAGE gel and HPLC analyses and the grade is analytic standard. Human recombinant TNF-α protein (Purity: 98%) was purchased from PeproTech (Rocky Hill, NJ, United States). Griess reagent (cat. no. G4410), Hoechst staining solution (cat. no. 94403), 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (cat. no. M5655), Corning trans-well Wright-Giemsa stain solution and 6-diamidino-2-phenylindole dihydrochloride (DAPI) (cat. no. D9542) were purchased from Sigma–Aldrich (St. Louis, MO, United States). p-ERK, ERK, p-p38, p-JNK, JNK and NF-kB were purchased from Cell signaling technology (Danvers, MA, United States). And, MMP-9, MMP-2, p38, β-actin, p-IkB and IkB antibodies were purchased from Santa Cruz Biotechnology (Paso Robles, CA, United States).

The breast cancer cell line MCF-7 acquired from ATCC (Rockville, MD, USA) were subcultured with Roswell Park Memorial Institute Medium (RPMI) 1640 media (Hyclone, Marlborough, MA, USA) containing 10% of heat inactivated (v/v) FBS (fetal bovine serum) (GIBCO BRL, Grand Island, NY, USA), 1 mM l-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a humidified atmosphere of 95% air and 5% CO₂. Human umbilical vein endothelial cells (EA.hy 926 cells, ECs) were obtained from ATCC and cultured in medium 199 (GIBCO BRL, Grand Island, NY, USA) supplemented with 20% FBS, 2 mM l-glutamine, 5 U/mL heparin, 100 IU/mL penicillin, 10 μg/mL streptomycin, and 50 μg/mL EC growth supplements. Antibodies against COX-2, MMP-2, MMP-9, VEGF, β-Catenin, Cyclin D1, C-myc, ICAM, E-cadherin, Survivin, IkB, p-IkB, and NF-κB were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibody against β-actin was from Sigma (Beverly, MA, USA). Peroxidase-labeled donkey anti-rabbit and sheep anti-mouse immunoglobulin, and an enhanced chemiluminescence (ECL) kit were purchased from Amersham (Arlington Heights, IL, USA). All other chemicals not specifically cited here were purchased from Sigma Chemical Co. (St. Louis, MO, USA). We used the anthocyanins isolated from Vitis coignetiae Pulliat [8 (link)].