Cell cultured and transfection
AML12, Hepa1-6 and HEK-293T cells were obtained from the Stem Cell Bank, Chinese Academy of Sciences (Shanghai, China). AML12 cells were cultured in DMEM/F-12 medium (Gibco) with 10% (v/v) fetal bovine serum (FBS) (Gibco), 1% (v/v) ITS liquid media supplement (Sigma), and 40 ng/ ml dexamethasone (Sigma). HEK-293T and Hepa1-6 cells were cultured in DMEM medium (Gibco) and RPMI-1640 medium (Gibco), respectively, with 10% (v/v) fetal bovine serum (FBS) (Gibco) and 1% penicillin/streptomycin. All cells were cultured under standard culture conditions of 37°C in a humidified atmosphere containing 5% CO2.
Hepa1-6 cells were transfected with 50 nM siRNA (RiboBio) and Lipofectamine™ RNAiMAX (Invitrogen). The negative control siRNA has no significant sequence similarity with mouse gene sequences. Si-lnc027912 sequence: GUGUAUUGCUUUGAGUUCATTUGAACUCAAAGCAAUCCACTT; si-control sequence: UUCUCCGAACGUGUCACGUTTAC GUGACACGUUCGGAGAATT.
Construction of overexpression cell line
After mixing the plasmids (pCDH-GFP-lnc027912 and pCDH-GFP) and pPACKH1-plasmid Mix, they were transfected into HEK-293T cells, the culture medium was collected, and the virus particles were precipitated with PEG-it. Puromycin was added to AML12 cells to screen out the cell line stably expressing lnc027912, and the concentration of puromycin in AML12-GFP or AML12- lnc027912 cells was 0.8 μg/ml.
Oleic acid (OA) and palmitic acid (PA) treatment, and Oil red O staining
The AML12-GFP and AML12-lnc027912 cells were seeded in 12 wells at the density of 5× 104 cells per well in DMEM/F-12 medium. After for 12 h, the cells were washed with 1×PBS for one time, then 200 μM OA (Sigma) or PA (Sigma) treatment for 24 h. All samples were fixed with 4% (wt/vol) paraformaldehyde in PBS, and refrigerated at 4°C overnight. Then, cells were stained with Oil red O, as previous described [18].
Measurement of triglyceride (TG) in hepatic cell
The AML12-GFP and AML12-lnc027912 cells were treated with OA (200 μM) and PA (200 μM) for 24 h, then samples were lysed with cell lysis buffer and obtained the liquid supernatant. The TG was measured according to the TG manufacturer’s protocols (Jiancheng Bioengineering Institute, Nanjing, China).
Quantitative RT-PCR
Total RNA was isolated from cells using the TRIzol reagent (Invitrogen). Remove genomic DNA with 4×gDNA wiper Mix, and then add 5×HiScriptⅡqRT SuperMixⅡ (Vazyme;) to synthesize cDNA. Quantitative RT-PCR was performed on the CFX96TM real-time PCR system (Applied Biosystems). The PCR reagent mix was obtained from Vazyme. The data was analyzed using the ΔΔCT method. GAPDH expression was an control to normalize the data. All primers sequences used are listed in the Table S1.
Western blot analysis
Cellular protein was extracted with cell lysis buffer and measured using an BCA Protein Assay Kit (Beyotime). Then, the proteins were separated via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Immunoblotting was performed using select primary antibodies, followed by probing with the recommended secondary antibodies, GAPDH was used as a control. Finally, the signals were detected using a ChemiDocTM Imaging System (Bio-Rad).The antibodies used are listed in the Table S2.
Immunofluorescence
AML12-GFP and AML12-lnc027912 cells were treated with OA and PA. Then, samples were fixed with 4% paraformaldehyde and further permeabilized with 0.5% Triton X-100. The specially primary (FAS and SREBP1C) and secondary (Alexa Fluor 555-labeled Donkey Anti-Mouse IgG(H+L), (Beyotime) and Cy3-labeled Goat Anti-Mouse IgG (H+L) , (Beyotime)) antibodies were incubated with the cells. The nuclei were stained with 4’,6-Diamidino-2-phenylindole (DAPI, Beijing Solarbio Science & Technology Co., Ltd. Beijing, China) . The images were captured under a laser confocal microscope (Nikon, Tokyo, Japan), and these pictures were further quantified using Image J software (National Institutes of Health, Bethesda, MD, USA).
Luciferase reporter assays
The SREBP1C promoter containing the far-upstream element sequence (about-2000-0 bp) was sub-cloned and inserted into the pGL3-basic vector (Promega, Madison, WI, USA). The recombinant plasmid (pGL3-SREBP1C-promoter vector) was obtained by MiaoLingBio. HEK-293T cells were seeded onto 48-well plate and transfected with pCDH-lnc027912, pGL3-SREBP1C, and phRL-TK vectors. After incubation for 36 h, the cells were treated with OA (200 μM) and PA (200 μM) for 24 h. Then, the cells were lysed in 1×passive lysis. A Dual-luciferase reporter assay system (Promega) was performed according the manufacturer’s protocols, with Renilla luciferase serving as a transfection control.
Statistical analysis
All experiments were repeated at least three times, and the results were presented as mean ± SD. Statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Software, San Diego, CA). The P values were calculated using a one-way ANOVA. A P value of <0.05 was considered statistically significant.