High fat diet d12492

All chemicals and reagents were obtained from Sigma, USA. High fat diet (D12492) was from Research Diets, Inc. NJ, USA. QRT-PCR primers were obtained from IDT, USA and antibodies were from Santa Cruz Biotechnology, USA.

All chemicals and reagents were obtained from Sigma, St Louis, MS, USA. Capsiate was obtained from Alamone Laboratory, Jerusalem, Israel. High fat diet (D12492) was obtained from Research Diets, Inc. New Brunswick, NJ, USA. qRT-PCR primers were obtained from IDT, Coralville, IA, USA and antibodies were from Cell signaling Technology, Danvers, MA, USA, and Santa Cruz Biotechnology, Dallas, TX, USA.

Rabbit anti-PPARγ, rabbit anti-ACC antibodies, and mouse anti-β-actin were purchased from Cell Signaling Technology (Beverly, MA, USA). Rabbit anti-fatty acid synthase, anti-death receptor Fas/CD95 antibodies, and mouse anti-SREBP1c were obtained from Abcam Inc. (Cambridge, MA, USA). Rabbit anti-active caspase-3 antibodies, and mouse anti-insulin were obtained from Servicebio (Wuhan, China). Goat anti-mouse FITC-conjugated IgG and DyLight 594 AffiniPure donkey anti-rabbit IgG were obtained from EarthOx LLC (San Francisco, CA, USA). The normal chow diet (D12450B) and the high-fat diet (D12492) were obtained from Research Diets, Inc. (New Brunswick, NJ, USA).

EGCG (purity > 95%) was obtained from Huzhou Rongkai Foliage Extract Co., Ltd. (Huzhou, China). A fatty acid-albumin complex solution containing palmitic acid (PA, Sigma–Aldrich, St Louis, MO, USA) and fatty acid-free bovine serum albumin (BSA, Sigma, St Louis, MO, USA) was prepared as described previously [44 (link)]. All cell culture reagents were purchased from Gibco (Grand Island, NY, USA). Primary antibodies specific for NF-κB, phospho-NF-κB, IκB-α, phospho-IκB-α, Stat3, phosphoStat3, Jak2, and phospho-Jak2 were purchased from Cell Signaling Technology (Beverly, MA, USA), and Iba-1 was obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). D12450J normal chow diet and D12492 high-fat diet were purchased from Research Diets, Inc. Co., Ltd. (New Brunswick, NJ, USA).

Male C57BL/6 mice were obtained from Charles River Laboratory Japan, Inc. (Yokohama, Japan). From the age of four weeks, mice were fed control diet (Oriental Yeast, Tokyo, Japan) or D12492 high-fat diet (Research Diets, NJ, USA) containing 60% fat. Animal studies were performed according to guidelines of the Animal Research Committee of Tokushima University, and were approved by the Animal Research Committee of Tokushima University. Mice were humanely sacrificed by cervical dislocation.
White adipose tissues were collected from epididymal fat pads, minced, and digested for 45 min at 37°C in PBS(-) supplemented with 2% bovine serum albumin (Sigma, MO, USA) and 2500 units/mL type II collagenase (Worthington, OH, USA). Digests were then incubated for 15 min in 10 mM EDTA, and passed through 250-μm nylon mesh. The filtrate was centrifuged at 1,200 rpm for 5 min at 4°C. Adipocytes and stromal vascular fractions were recovered from the supernatant and pellet, respectively.

For Experiment 1, 90 C57BL6/J mice, 49 male and 41 female mice weaned from our in-house breeding colony were used. Our in-house breeding colony founders were obtained from Jackson Laboratories (Bar Harbor, ME, USA). For Experiment 2, 16 eleven-week-old male NCI C57BL6/NCr mice were obtained from Charles River Laboratories NCI (Frederick, MD, USA). These inbred strains of C57BL6 mice were fed a purified high fat diet (60% dietary Kcal as fat) obesogenic diet formulation (D12492 high fat diet; Research Diets, Inc., New Brunswick, NJ [12 ,13 ]). Mice were maintained on a 12 h light/dark cycle at 27.5 ± 2 °C with 30% relative humidity and given ad libitum access to diet and distilled water until they were randomized to experimental groups. All animal studies were performed in accordance with the Colorado State University Institutional Animal Care and Use Committee, protocol 18-7746.