Fatostatin

The following antibodies and reagents were used: Anti-SREBF1 (Proteintech, 14088-1-AP, 1:1000 for western blotting and 4 μg for ChIP), anti-KLF5 (Santa Cruz Biotechnology, sc-398409X, 1:1000 for western blotting, and 4 μg for ChIP), anti-TP63 (R&D Systems, AF1916-SP, 1:1000), anti-Actin (Santa Cruz Biotechnology, sc-8432, 1:2000), anti-ACLY (Cell Signaling Technology, 4332, 1:1000), Anti-FASN (Cell Signaling Technology, 3180, 1:1000), anti-GAPDH (Cell Signaling Technology, 2118, 1:2000), anti-mTOR (Cell Signaling Technology, 2972S, 1:1000), anti-Phospho-mTOR (S2448) (Cell Signaling Technology, 5536T, 1:1000), anti-Phospho-MEK1 (Ser298) (Cell Signaling Technology, 9128S, 1:1000), anti-MEK1/2(D1A5) Rabbit (Cell Signaling Technology, 9124S, 1:1000), anti-p70 S6 Kinase (Cell Signaling Technology, 9202S, 1:1000), anti-Phospho-p70 S6 Kinase (Cell Signaling Technology, 9205S, 1:1000), anti-mouse IgG-HRP (Jackson ImmunoResearch Laboratories, Inc., 115-035-003, 1:10000), anti-rabbit IgG-HRP (Jackson ImmunoResearch Laboratories, Inc., 111-035-144, 1:10000), anti-goat IgG-HRP (Jackson ImmunoResearch Laboratories, Inc., 705-035-003, 1:10000), HCS LipidTOX™ Green Neutral Lipid Stain (Thermo Scientific, H34475, 1:100), Lipofectamine RNAiMAX (Thermo Scientific, 13778150), and Fatostatin (Cayman Chemical, 12562).

Six‐week‐old BALB/c male mice (n = 20) were used in the present study. A 14:10 h light/dark photocycle was used, and the supplementation of food and water was conducted in accordance with the instructions of Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University. Food was replenished and the cage was cleaned once a week, and the water supply was provided via a nozzle in each cage set into a shelf. With a floor area of 451 cm², each cage contained five mice, in accordance with the instructions of National Research Council [16 (link)]. All mice were intraperitoneally injected with 30 µL of K/BxN mouse serum, as described previously [13 (link), 14 (link)]. The injection day was designated as day 0. Mice were then divided into two groups: control and fatostatin‐treated, and each group involved 10 mice. Mice from the control group were administered 80 µL of DMSO (#043‐07216; FUJIFILM Wako Pure Chemicals Corp, Osaka, Japan), whereas those from the fatostatin group were injected with 0.6 mg of fatostatin (#13562; Cayman, Ann Arbour, MI, USA) in 80 µL of DMSO. Both groups were administered their respective injections for 3 days. To reduce suffering, intraperitoneal injections were performed as infrequently as possible.