Ago mir 34a

The human L02^{46 (link)} hepatic cell line was obtained from China Cell Culture Center (Shanghai, China). The cells were grown in 1640 containing 10% (v/v) fetal bovine serum (FBS) in a humidified incubator with 5% CO2 at 37 °C. Both 1640 and FBS are Invitrogen products that were purchased from Life Biotechnologies (Carlsbad, CA, USA). Cholic acid (98% purity) and palmitic acid (PA; 98% purity) were purchased from Sigma-Aldrich (St. Louis, MO, USA). When indicated, cells were treated with 10 μM CA, 10 μM Cholic acid, or neither (control) for 12 h before processing for total protein and RNA extraction.^{32 (link)} For the experiments, L02 cells were transfected with 10 nM ago-miR-34a (GenePharma, Shanghai, China) or ago-miR-negative control using Lipofectamine 2000 (Invitrogen). After 24 h, the cells were incubated without (control) or with 10 μM CA. Hepatocytes were harvested at 36 h post transfection and processed for total RNA and protein extraction. To induce lipotoxicity and to establish the cell model, cells were incubated with 0.5 mM PA for 24 h.^{47 (link)}

miRNA antagomir or agomir is a type of specially labeled and chemically modified miRNA. Antagomir is special for inhibiting the expression of endogenous miRNA. Agomir can regulate the biological function of target gene by mimicking endogenous miRNA. Antagomir-34a, agomir-34a, or matched negative controls (antagomir-NC, agomir-NC) was purchased from GenePharma (Shanghai, China). CREB short hairpin RNA (CREB-shRNA) (Oligobio, Beijing, China) was constructed to specifically block CREB according to the manufacturer's instructions. The cDNA sequences encoding CREB protein were subcloned into the pcDNA3.1-HA vector (Youbio, Hunan, China) to generate the CREB expression plasmid. Cell transfection was performed with Lipofectamine 2000 Reagent (Invitrogen, USA), as per the manufacturer's manuals.