Linoleic acid

For Oil Red O staining, linoleic acid (Wako) was added into the medium at 10 mg/ml and incubated for 30 minutes. After washed with PBS, cells were fixed with 4% PFA at room temperature for 30 minutes and washed with 60% isopropanol, followed by incubation for 30 minutes with Oil Red solution which Oil Red powder (Sigma Aldrich) was dissolved into isopropanol and diluted to 60% with distilled water. Then cells were washed with distilled water three times. For PAS staining, cultured cells were stained by periodic acid-Schiff (MUTO PURE CHEMICALS) following the manufacturer’s instructions. For paraffin-embedded spheroids, the same procedure was carried out after deparaffinization. For ICG uptake assay, indocyanine green (Wako) was added in the medium at 0.1 mg/ml and incubated for 1 hour, followed by washing with PBS three times.

BV2 cells were cultured in either DMEM (high glucose; Wako) or DMEM/nutrient mixture F-12 (DMEM/F12; Thermo Fisher Scientific) containing 10% fetal bovine serum (FBS), 100 units penicillin and 100 mg streptomycin (P/S; Thermo Fisher Scientific). For screening, BV2 cells were cultured in DMEM with 10% FBS and P/S in the presence or absence of 150 μM L-alanine (Wako), 50 μM L-glutamic acid (Wako), 50 μM L-aspartic acid (Wako), 50 μM L-asparagine (Wako), 150 μM L-proline (Wako), 0.0052 pM CuSO₄ (Wako), 1.5 pM FeSO₄ (Wako), 1.5 pM ZnSO₄ (Wako), 150 nM linoleic acid (Wako), 500 nM lipoic acid (Sigma), 15 nM biotin (Sigma), 500 nM vitamin B12 (Wako), and 15 μM hypoxanthine (Sigma). Pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid tetrasodium salt (PPADS) was purchased from Alexis Corporation. Dipyridamole (DY) and allopurinol (ALP) were purchased from Tokyo Chemical Industry. Hoechst 33342 was purchased from Thermo Fisher Scientific. DAPI was purchased from Dojindo. FAT3 siRNAs (NM_001080814, siRNA ID; SASI_Mm02_00293873) and MISSION siRNA universal negative control (SIC001) were purchased from Sigma; 50 nM FAT3 siRNAs were transfected using Lipofectamine RNAi Max Reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions.

Sodium polysulfides (Na₂S_n) were purchased from the DOJINDO chemical laboratory, Japan. Human albumin 25% “BENESIS” was provided by the Japan Blood Products Organization (Tokyo, Japan). Spectra pore 7 (MWCO: 3500) as a sulfur-free dialyzed membrane, was purchased from Repligen, USA. Ascorbic acid, dithiothreitol (DTT), zinc acetate dihydrate, LabAssay™ Creatinine, and linoleic acid were purchased from FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan. The BCA protein assay kit and BUN Colorimetric Detection Kit were obtained from Thermo Fisher Scientific, Waltham, MA, USA. Other chemicals were of the best grades commercially available, and all solutions were made using deionized water.

Lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, and glutaraldehyde were purchased from Tokyo Chemical Industry Co. Ltd. (Tokyo, Japan). Caprylic acid, capric acid, linoleic acid, SLS, sodium chloride (NaCl), disodium hydrogen phosphate 12-water (Na₂HPO₄•12H₂O), potassium dihydrogen phosphate (KH₂PO₄), sodium chloride (KCl), potassium hydroxide (KOH), hydrochloric acid (HCl), manganese (II) chloride tetrahydrate (MnCl₂•4H₂O), magnesium sulfate heptahydrate (MgSO₄•7H₂O), iron (II) sulfate heptahydrate (FeSO₄•7H₂O), calcium chloride dihydrate (CaCl₂•2H₂O), agar, crystal violet, Hank’s balanced salt solution (HBSS)(+) and 99.5% ethanol (EtOH) were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). SLES was purchased from the NOF Corporation (Tokyo, Japan). The Dulbecco’s phosphate-buffered saline (d-PBS)(+) preparation reagent (with Ca and Mg) (100×) and fetal bovine serum (FBS) were purchased from Nacalai Tesque (Kyoto, Japan) and Thermo Fisher Scientific K.K. (Tokyo, Japan), respectively. WELPAS^® antiseptic solution for hands (0.2%), an alcohol-based disinfectant, was purchased from Maruishi Pharmaceutical Co. Ltd. (Osaka, Japan).

All the chemicals used for this study were of analytical grade. Linoleic acid (18:2 cis-9,12), oleic acid (18:1 cis-9), anhydrous pyridine, ethanol, methanol, and formic acid were purchased from FUJIFILM Wako Pure Chemical Corporation (Kyoto, Japan). Acetonitrile was purchased from Nacalai Tesque (Kyoto, Japan).