Mcd diet

Seven-week-old male C57BL/6J mice were obtained from Samtaco Korea (Seoul, Korea). The mice were given free access to water and kept at a constant room temperature under a 12/12-hour light/dark cycle. They were allowed to adapt to their food and environment for 1 week before starting the experiment. The C57BL/6 mice were divided into 5 groups (7 mice per group) and its group shown in Table 1. Namely, normal diet (Dyets Inc., Bethlehem, PA, USA), MCD diet (Dyets Inc., Bethlehem, PA, USA), SIL 100 (MCD diet supplemented with silymarin 100 mg/kg/day), BDMC 100 (MCD diet supplemented with BDMC 100 mg/kg/day), SIL 50+BC 50 (MCD diet supplemented with silymarin 50 mg/kg/day + BDMC 50 mg/kg/day) was orally administered by gavages to the mice daily during the 4 weeks of diet feeding. The composition of the experimental diet was shown in Table 2. After four weeks, animals were sacrificed via CO₂ inhalation for the collection of blood and liver samples. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication No. 85–23, revised 1996) and was approved by the Institutional Animal Care and Utilization Committee for Medical Science of Wonkwang University (Approval no.WKU-15-100).

8 week-old Wild-type and PTPRO KO Mice were fed with a Normal diet or MCD diet (Dyets, Bethlehem, PA, USA; #518810) for 4 weeks or Western diet [WD: purchased from Dyets, Inc. Wuxi, China) for 16 weeks. A total of 6 mice were in each group. These mice were euthanized after feeding with MCD diet for 4 weeks or WD diet for 16 weeks and had been confirmed to develop the histopathological features of NASH, such as steatosis, inflammation, and fibrosis.

Eight-week-old male wild-type C57BL/6 mice (SLAC Laboratory Animal Co. Ltd., Shanghai, China), with weights of 24–25 g, were allowed to adapt to housing in the Laboratory Animal Center of Wenzhou Medical University for 1 week prior to random assignment to experimental cohorts (room temperature: 22 ± 2 °C; humidity: 45 ± 5%). After one week of acclimatization with a standard normal-chow (NC) diet and tap water ad libitum, all animals were randomly assigned to three experimental groups (n  =  6/group): NC, MCD diet (Dyets Inc., Bethlehem, PA, USA), and MCD diet with 0.6% D-PUFAs (Retrotope Inc., Los Altos, CA, USA). The diet ingredients are listed in Supplementary Table S1. The component of the D-PUFAs was chosen according to previous mice studies [12 (link),15 (link),16 (link)]. After feeding for 5 weeks, all mice were sacrificed after deep anesthesia by a peritoneal injection of pentobarbital sodium (50 mg/kg), and then the blood and the liver tissues were harvested.
The animal procedures were performed according to institutional guidelines, and this study was approved by the Wenzhou Medical University Animal Experiment Committee (wydw2019-0945), in accordance with the 3Rs policy.

Male C57Black6J/jcl mice, aged 8 weeks, were purchased from CLEA Japan (Shizuoka, Japan). Mice were studied at 2 and 4 weeks after MCD diet (Dyets, Bethlehem, PA, USA) feeding or normal diet feeding (control group). They were allowed free access to chow and tap water and housed in a temperature-controlled room maintained on a 12-h light/dark cycle with lights on at 8:00 am. The experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee of Shionogi Research Laboratories, Osaka University Graduate School of Medicine, and Animal Care Committee of Chiba University.