Lutein

The human ARPE19 cell line was maintained in Dulbecco’s modified Eagle’s medium (DMEM) and Ham’s nutrient mixture F-12 medium (Gibco, Carlsbad, CA) supplemented with 10% foetal bovine serum (FBS), penicillin (100 units/ml) and streptomycin (100 μg/ml) at 37 °C under a humidified atmosphere with 5% CO₂. Lutein-rich marigold extract containing 92% Lutein (Wakasa Seikatsu, Co. Ltd.) was emulsified as described previously30 (link). Briefly, Lutein was first diluted in a solution of ethanol (Wako Pure Chemical, Osaka, Japan) and Tween 80 (Sigma-Aldrich) (310 μl:10 μl)30 (link). The ethanol was evaporated off and a micelle emulsion was formed using nitrogen gas; this was diluted in DMEM prior to addition to the culture medium30 (link). The culture medium was removed and serum-free medium was added before ARPE19 cells were treated with 25 μM Lutein, 50 μM Lutein, or vehicle for 3 h. The cells were then washed with 1 ml PBS, sonicated in an ice bath and centrifuged at 10,000 g for 5 min at 4 °C prior to measuring the supernatant SOD activity, or placed in TRIzol reagent (Life Technologies) for mRNA extraction and real time RT-PCR analyses (see above).

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.

All chemicals except for the standard of chlorophyll b, trans-β-apo-8′-Carotenal, and lutein were purchased from Fujifilm Wako Pure Chemical (Osaka, Japan). Standards of chlorophyll b and trans-β-apo-8′-carotenal were purchased from Sigma-Aldrich (Tokyo, Japan). lutein standard was purchased from EXTRASYNTHESE S.A. (Genay, France).