After 20 weeks on diet, by which time liver inflammation is established, mice in each group were randomly split into treatment arms, either with drug (TTP488, Selleckchem, Houston, TX), dissolved as per manufacturer recommendations in 10% DMSO, 40% PEG300, 5% Tween 80, and 45% ddH20, or vehicle alone. Mice received daily intraperitoneal (i.p.) injections of drug (4 mg/Kg) or equivalent volume of vehicle for 30 days, while they continued to be on their respective diets.
Growth protocol
Nine-week-old C56BL/6J male mice were purchased from Jackson Laboratory (Bar Harbor, ME) and were acclimated for 3 weeks, during which all mice received standard rodent chow diet. Mice had unrestricted access to food and water and were housed in standard pathogen-free facilities with 12:12-h day-night circadian cycles. Adult male mice were assigned to 2 groups of similar starting weight, to receive chow, or FFC diet, which provides 40% kcal from fat and is composed of 34% sucrose, 20% milk fat, and 0.21% cholesterol (AIN-76A Western Diet, originally manufactured as D12079B, TestDiet, St. Louis, MO). Mice in the FFC group also received sweetened drinking water (23.1 g/l fructose and 18.9 g/l glucose) ad lib. This murine model results in the development of insulin resistance, adipose tissue inflammation, and NASH with hepatocyte ballooning and fibrosis, displaying high fidelity to human NASH.
Extracted molecule
total RNA
Extraction protocol
Liver tissue was dissociated with the mouse liver dissociation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) and the gentleMACS Dissociator (Miltenyi Biotec) following manufacturer’s protocols. The enzymatically digested liver was passed through a 40 μm filter to remove debris and cell clusters. The filtered dissociate was centrifuged and IHLs were isolated by Percoll-density gradient centrifugation. Isolated RNA quantity and quality were assessed with a NanoDrop ND1000 (ThermoScientific, Waltham, MA).
Label
biotin
Label protocol
100 ng of each total RNA sample was labeled according to the manufacturer's instructions.
Hybridization protocol
Hybridization was done according to the manufacturer's instructions.
Scan protocol
Gene expression was quantified on the nCounter Digital Analyzer (NanoString Technologies).
Description
Gene expression of IHLs from FFC fed mouse treated with TTP488.
Data processing
Raw data was normalized in the nSolver Analysis Software (NanoString Technologies) by the geometric mean of 10 housekeeping genes and 6 positive controls.
Nanostring of gene expression in intrahepatic leukocytes from mouse fed a chow or high-fat, -fructose, and -cholesterol (FFC) diet and treated with vehicle or a pharmacological inhibitor of the receptor for advanced glycation end products (RAGE), TTP488
