α tocopherol

AF cells were treated with H₂O₂ (0, 10, 100 μM) (Wako, Tokyo, Japan), buthionine sulfoximine (BSO; 0, 0.2, 1 mM) (Sigma-Aldrich), and TNFα (50 ng/ml) (R&D Systems, Minneapolis, MN, USA) for 24 hours. For analysis of phosphorylation, AF cells were treated with 100 μM H₂O₂ for 0, 5, 10, 15, 30, or 60 minutes and 50 ng/ml TNFα for 10 minutes. Cells were pretreated with MAPK signaling inhibitors for 30 minutes followed by incubation with H₂O₂ or BSO for 24 hours. In this assay, we utilized p38 inhibitor (SB203580, 10 μmol/l), JNK inhibitor (SP600125, 10 μmol/l), or ERK inhibitor (PD98059, 10 μmol/l). All inhibitors were purchased from Wako. To study the effect of antioxidative agents, H₂O₂-treated, BSO-treated, or TNFα-treated AF cells were cultured with 100 μM NAC and 20 mM α-tocopherol (Wako) solved in ethanol for 15 minutes, 30 minutes, or 24 hours. In the case of α-tocopherol experiment, ethanol was added at the same concentration to ROS-mediated or TNFα-treated AF cells. The experiment of α-tocopherol treatment was independently carried out five times, and all other experiments were independently carried out three times.

LPS and antibodies against β-actin were purchased from Sigma-Aldrich (USA), and lutein, β-carotene, and α-tocopherol were obtained from Wako Pure Chemicals (Japan). Zeaxanthin was purchased from Cayman Chemical Co. The reagents used in this study are shown in Supplementary Table S2.
Antibodies against p38 (#9212), phospho-p38 (#4631), JNK (#9258), phospho-JNK (#4668), Erk1/2 (#9102), phospho-Erk1/2 (#4370), IκB (#4814), phospho-IκB (#2859), NF-κB p65 (#8242), phospho-NF-κB p65 (#3033), HIF-1α (#36169), Caspase-1 (#24232), Cleaved Caspase-1 (#89332), NLRP3 (#15101), ASC (#67824), and AIM2 (#63660) were purchased from Cell Signaling Technology, USA. The antibodies used in this study are shown in Supplementary Table S3.