Myriocin

C57BL/10SnJ or C57BL/10ScSn-Dmd^mdxJ mice were purchased from the Janvier and Jackson Laboratories. Myriocin (Enzo Life Sciences, Farmingdale, NY) was first dissolved in DMSO and then mixed with phosphate-buffered saline (PBS). Three-week-old mice were treated with Myriocin for 6 months at a dose of 0.4 mg/kg or with corresponding volume of DMSO three times per week. The dose was selected on the basis of previous literature showing that Myriocin (0.3 mg/kg) every other day reduced atherosclerosis (56 (link)). For stock solution, 20 mM Myriocin was dissolved in DMSO. Another cohort of 3-week-old mice was treated for 2 months with Myriocin and/or deflazacort. Deflazacort [1 mg/kg intraperitoneal injections once per week] was administered as described in (50 (link)). All mice were housed in microisolator cages in a room illuminated from 7:00 a.m. to 7:00 p.m. with ad libitum access to normal chow diet and water. All the animal experiments are authorized by the local animal experimentation committee of the Canton de Vaud, Switzerland, under license 2890.1.

BMDMs were isolated from the femurs and tibias of mdx mice by first cleaning the bones with a scalpel and 70% ethanol, followed by cutting the bones at the ends of the marrow and centrifugation at 20,000g for 15 min in 1.5-ml Eppendorf tubes. After removing fibroblasts by taking the supernatant after 24 hours, cells were pooled, counted, and plated at 1.6 million cells/ml in six-well plates with macrophage growth medium consisting of 85% complete RPMI 1640 [RPMI medium (Gibco), 10% FBS, and Hepes] and 15% L929-conditioned medium for 6 days. BMDMs were then stimulated with IL-4 (100 ng/ml) for 48 hours in the presence of 10 μM myriocin or corresponding volume of DMSO. A stock solution of 20 mM in DMSO was used for myriocin (Enzo). For sphingolipid measurements, four to six wells of macrophages were pooled as one replicate.

Wild type mice (C57BL/6J, Charles River Laboratories, Wilmington, MA) and CerS2^+/− mice (obtained from Drs. A. Futerman and S. Summers) were used as the source of PMH and were isolated as previously described [46 (link)]. PMH were treated with PA, MA or OA alone or in combination at equimolar concentrations (0.25-1mM) for 1-12 hours. Stock solutions of PA, MA or OA were prepared as described [6 (link)] and the final working concentrations were made with 0.5-1% (w/v) fatty acid free bovine serum albumin (BSA, Roche). FFA:BSA ratio ranged from 3:1 to 6:1 depending on the final FFA concentration. High FFA:BSA ratio has been observed in states of insulin resistance and obesity, two risk factors for NASH [47 (link)]. In some cases, PMH were treated with butylated hydroxyanisole (BHA, 50 - 200 μM) (Sigma-Aldrich) in absolute ethanol, z-VAD-FMK (5 - 15 μM) (Abcam, Cambridge, UK) in dimethyl sulfoxide (DMSO), myriocin (20 μM) (Enzo Life Sciences, Farmingdale, NY) in NaOH (50 mM) and DMSO (0.1%), tauroursodeoxycholic acid (TUDCA, 25 μM) (Calbiochem, Billerica, MA), ruthenium red (RR, 10 μM) and the JNK inhibitor SP600125 (20μΜ). Cell death, lipodomic analyses, total ceramide levels, DES activity from NBD-dihydroceramide C12; ROS generation and determination of ER stress are described in Supplemental Material section.