Glutamine

Human RPE cells (h1RPE7 cells) [9] (link), were purchased from the European Collection of Cell Cultures (Salisbury, UK) and were grown in Ham's F10 medium (Gibco^®, Life Technologies, Carlsbad, CA) containing 20% (v/v) fetal calf serum, 2 mM glutamine (Nacalai tesque, Kyoto, Japan) and 1 μg/mL puromycin (Gibco^®). For the in vitro AMD model, h1RPE7 cells were treated with 300 μg/mL of AGE-bovine serum albumin (BSA) (Calbiochem^®, Merck KGaA, Darmstadt, Germany) and 20 μM HQ (Wako Pure Chemical Industries, Ltd., Osaka, Japan) as described [8] . ARPE-19 cells were grown in 1:1 mixture of Dulbecco's modified Eagles medium (Gibco^®) and Ham's F12 medium (Gibco^®) containing 10% (v/v) fetal calf serum, 100 units/mL penicillin G (Wako), and 100 μg/mL streptomycin (Wako) as described [8] .

The B16-F0 mouse melanoma cell line (cat. no. JCRB0202) was purchased from Japanese Collection of Research Bioresources (JCRB) Cell Bank and maintained in Eagle's Minimum Essential Medium (EMEM; FUJIFILM Wako Pure Chemical Corporation) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich; Merck KGaA), 100 U/ml penicillin and 100 µg/ml streptomycin (both from Nacalai Tesque, Inc.) under mycoplasma-free conditions. Clone M3 (Cloudman S91) melanoma cell line was obtained from European Collection of Authenticated Cell Cultures (ECACC) and cultured in Ham's F10 medium (FUJIFILM Wako Pure Chemical Corporation) supplemented with 15% FBS, 2 mM glutamine (Nacalai Tesque, Inc.), 100 U/ml penicillin and 100 µg/ml streptomycin. 293T and 293FT cells were obtained from Invitrogen; Thermo Fisher Scientific, Inc. HEK-Blue™ TGF-β cells were purchased from InvivoGen. 293T, 293FT and HEK-Blue™ TGF-β cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Nacalai Tesque, Inc.) supplemented with 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin. The cultured medium for 293FT cells was also supplemented with 1% non-essential amino acid solution (Nacalai Tesque, Inc.). All cell lines were cultured in a humidified incubator containing 5% CO₂ at 37°C.

Normal human dermal fibroblast (NHDF) was purchased from Lonza Co. Ltd. (Tokyo, Japan). Fibroblasts were grown in Dulbecco’s modified Eagle’s medium (DMEM, Nacalai Tesque, Kyoto, Japan) supplemented with 2% fetal bovine serum (FBS; MP Biomedicals, Illkirch, France), 2 mM glutamine, 100 units/ml penicillin and 100 mg/ml streptomycin (Nacalai Tesque). The cells were incubated under 37°C in a humidified atmosphere of 5% CO₂ and 95% air in a CO₂ incubator. After 24 h, culture medium was replaced by the DMEM containing 2% FBS and P40, and the cells were subsequently pre-cultured for 1 week in the CO₂ incubator until the experiments.