Mouse liver cell line AML-12 was purchased from Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China) and cultured in DMEM-F12 medium containing 10% fetal bovine serum (FBS). Upon cell adherence, they were passaged the second day, and those in the logarithmic growth phase were used in the experiments.
AML-12 cells were stimulated by 100 mM ethanol for 24 h alone, or stimulated by 100 mM ethanol for 24 h, followed by transfection of siRNA-NEAT1 negative control (NC), siRNA-NEAT1, miR-129-5p mimic NC, miR-129-5p mimic, NEAT1 overexpression vector (overexpressed (oe)-NEAT1) together with miR-129-5p mimic NC, or oe-NEAT1 together with miR-129-5p mimic. The siRNA-NC, siRNA-NEAT1, mimic-NC, miR-129-5p mimic and oe-NEAT1 were all acquired from GenePharma Co., Ltd, (Shanghai, China).
AML12 cells were seeded in 12-well plates and cultured for 24 h before transfection. The transfection was conducted by Lipofectamine 2000 reagent (Invitrogen Inc., Carlsbad, CA, USA) when the cell confluence reached 70–90%. Transfected for 6 h, the medium was changed and cells were stimulated by 100 mM ethanol for 24 h. Finally, cells and cell supernatant were collected for the subsequent cell experiments.
AML-12 cells were stimulated by 100 mM ethanol for 24 h alone, or stimulated by 100 mM ethanol for 24 h, followed by transfection of siRNA-NEAT1 negative control (NC), siRNA-NEAT1, miR-129-5p mimic NC, miR-129-5p mimic, NEAT1 overexpression vector (overexpressed (oe)-NEAT1) together with miR-129-5p mimic NC, or oe-NEAT1 together with miR-129-5p mimic. The siRNA-NC, siRNA-NEAT1, mimic-NC, miR-129-5p mimic and oe-NEAT1 were all acquired from GenePharma Co., Ltd, (Shanghai, China).
AML12 cells were seeded in 12-well plates and cultured for 24 h before transfection. The transfection was conducted by Lipofectamine 2000 reagent (Invitrogen Inc., Carlsbad, CA, USA) when the cell confluence reached 70–90%. Transfected for 6 h, the medium was changed and cells were stimulated by 100 mM ethanol for 24 h. Finally, cells and cell supernatant were collected for the subsequent cell experiments.