Lipofectamine rnaimax transfection kit

For siRNA-mediated β-catenin gene silencing, we transfected HepG2 cells with 5 nM of β-catenin-specific siRNA or Stealth siRNA negative control, obtained from Dharmacon (Lafayette, Colorado, USA), using Lipofectamine RNAiMAX transfection kit (13,778–075; Invitrogen, MA, USA) according to the manufacturer's instructions. After transfection, cells were cultured under normal growth conditions (37 °C, 5% CO2) for 24 h without antibiotics. The silencing efficiency was checked by quantifying the expression of β-catenin with qRT-PCR. For GLP-1R gene silencing, we used the Dicer-Substrate Short Interfering RNAs (DsiRNAs) and TriFECTa^® Kits (http://www.idtdna.com/calc/analyzer) and the Lipofectamine RNAiMAX transfection kit (13778-075; Invitrogen, MA, USA) to transfect HepG2 cells with 20 nM of GLP-1R specific siRNA or negative scrambled siRNA, according to the manufacturer's instructions. The DsiRNAs-TriFECTa^® kit contains three Dicer-substrate 27-mer RNA duplexes specific for a single target gene. A pool of the three duplexes was used to silence GLP-1R. After the silencing of GLP-1R, a qRT-PCR was performed for the following genes: PPARγ, FAS, DGAT1, DGAT2, and ACC. We normalized the data to β-actin as an internal control and used the comparative 2-ΔΔCT method to calculate the relative expression.

Before transfection, TE-1 and KYSE30 cells were seeded in 6-well plates to approximately 60% confluence. siRNA, miRNA mimics and inhibitors were transfected using a lipofectamine RNAiMAX transfection kit (Invitrogen, USA), and overexpression plasmids were transfected using a lipofectamine3000 transfection kit (Invitrogen, USA) following the manufacturer’s instructions.

LSCs were transfected with 10 nmol/L of siRNA targeting Gli1 or Gli3 genes (listed in Table 1; Stealth RNAi™ siRNA duplexes, Invitrogen) for 48 hours using the Lipofectamine RNAi MAX transfection kit according to the manufacturer's protocol (Invitrogen, USA). Scramble siRNA (sc‐37007, Santa Cruz Biotechnology) that did not specifically target any gene were used as control.
To evaluate the influence of Gli1 and Gli3 knockdown on LSC proliferation, LSCs (2 × 10³/well) were seeded in 96‐well cell culture plate (Costar; cat. no. 3599) one day before transfection. Forty‐eight hours after siRNA transfection, cells were maintained in serum‐free DMEM/F12 for 2 hours and then stimulated by 44‐mer (10 µmol/L) for 24 hours. The level of LSC proliferation was evaluated by Cell Proliferation Assay Kit based on the amounts of nuclear dye binding (BioVision; Catalog # K307‐1000), according to the company's instruction. All assays were performed in triplicate, and the experiment was independently performed for three times.