2.1. Chemicals
Palmitic acid (PA) and stearic acid (SA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Stock solutions of PA and SA were prepared as described previously. The Gm12664-201 small interfering RNA (siRNA sequence: 5’‐CCCTCTTCTTTGAATTCAA‐3’) and the corresponding negative control were synthesized from RiboBio (Guangzhou, China).. For the Gm12664-201 overexpression assay, a pcDNA3.1 vector was designed to specifically express the open reading frame of Gm12664-201 containing full-length 3’-UTR and purchased from GENERAY Biotechnology (Shanghai, China).
2.2. Animals
8-weeks male C57BL/6 mice were purchased from Vital River Laboratories (Beijing, China). After 1-week adaptation, the mice were randomly divided into two groups and supplied with either a normal diet (ND, 15% fat) or a high fat diet (HFD, 36% fat) for 10 weeks. Food intake and body weight were recorded daily and weekly, respectively. At the end of the experiment, all mice were sacrificed and blood samples and livers were collected and stored at -80 ℃ for further use. The study was approved by the Harbin Medical University Institutional Animal Care Committee and performed in accordance with the Harbin Medical University guidelines for the care and use of laboratory animals.
2.3. Cell culture, treatments and transfection
Alpha mouse liver-12 hepatocytes (AML-12, ATCC Cat# CRL-2254, RRID: CVCL_0140) were purchased from the ATCC, and cultured in a 1:1 mixture of Dulbecco’s modified Eagle’s medium and Ham’s F-12 medium (DMEM/F12, SH30023.02, HyClone Laboratories. USA) with 0.005 mg/ml insulin (A189560, Gibco, USA), 40 ng/ml dexamethasone (D4902-25MG, Sigma-Aldrich) and 10% fetal bovine serum (G11-70500, Genial Biological Inc., USA), at 37℃ in a humidified 5% CO2 incubator. Cells were treated with PA, or SA at different times and doses to determine the most appropriate intervention conditions.
AML-12 cells were transfected with 100 nM Gm12664-201 siRNA /negative control siRNA or 2.5 mg per well (6 wells plate) Gm12664-201 overexpression plasmid using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocols. After 24-hours transfection, the cells were harvested for further assays or treated with SA or PA for further 24 hours.
2.4. RNA isolation and qRT-PCR analysis
The total RNA was isolated from liver tissues or cultured cells using TRIzol reagent (15596026, Invitrogen, USA), then reversed (High Capacity cDNA Reverse Transcription Kits, Applied Biosystems, USA) and quantified by qRT-PCR (7500 Real-Time PCR System, Applied Biosystems, USA). For Gm12664-201 and mRNA detection were performed using SYBR Green PCR Master Mix (Applied Biosystems, USA). The quantification data were normalized to β-actin expression. The primers were purchased from Sangon (Shanghai, China) and corresponding sequences were listed in table 1.
2.5. Oil red O staining
After the AML-12 cells were transfected with plasmids or siRNA and further cultured with SA for 24 hours, remove and discard culture medium, rinse the cell layer with PBS to remove remain medium. The cells were fixed with 4% paraformaldehyde for 20 minutes, followed by briefly rinsed with 60% isopropyl alcohol and stained with Oil Red O (Sigma-Aldrich) for 30 minutes, and counterstained with Mayers hematoxylin (Sigma-Aldrich) for 1 minute. The digital images were captured using a Nikon EclipseTi-S microscope (Nikon, Tokyo, Japan).
2.6. Triglyceride detection
Triglyceride (TG) deposits in the serum were measured using a Triglyceride test Kit (ApplyGen, Beijing, China, E1013) according to the manufacturer’s instructions. Intracellular TG contents were detected using kits obtained from Solarbo Technologies Inc (Beijing, China).
2.7. Western blot analysis
Western blot was done as described previously. All the primary antibodies (Cell Signaling Technology Cat# 3177, RRID: AB_2119845; Cell Signaling Technology Cat# 4970, RRID: AB_2223172) were diluted by 1:1000 according to the western blotting protocol. Alkaline phosphatase (AP) enzyme conjugated secondary antibody (Promega Cat# S3731, RRID: AB_430872) was applied with a working dilution of 1:7,500 as suggested.
2.8. Statistical analysis
All data are expressed as the mean ± standard deviation (SD) of at least three independent experiments. Comparisons between groups were analyzed using Student's t-test. The statistical analyses were performed using the GraphPad Prism (GraphPad Prism, RRID: SCR_002798). A two-sided P value < 0.05 was considered statistically significant.