Lipofectamine

Transient transfection of luciferase reporter plasmids was performed using lipofectamine 2000 (Invitrogen), according to the manufacturer’s instructions. Cells were plated in 12-well tissue culture plates at 2 × 10⁵ per well for overnight. Nucleic acid-lipofectamine 2000 mixtures containing 0.5 μg of luciferase reporter vectors, 0.5 μg of pSV-β-galactosidase control vector (Promega) and 0.5 μg of expressing vectors or 40 nM siRNA were exposed to cells. After 24 h, cell lysates were harvested and assayed for the Luciferase activity assay (Promega), performed according to the protocol recommended by the supplier. To normalize the transfection efficiency, the same cell lysates were also assayed for β-galactosidase activity using the β-Galactosidase enzyme assay (Promega).

DEF cells were co-transfected with 400 ng/well of pcaggs-UL47-Flag or an empty vector, 400 ng/well of reporter plasmid IFN-β-Luc, and 4 ng/well of pRL-TK (Promega, USA) using Lipofectamine 3000, and the cells were stimulated with 50 ng/ml poly(I:C) at 12 h after transfection and harvested at 24 h after stimulation. Finally, we detected the firefly luciferase activity by the dual-luciferase assay system (Promega, USA) according to the manufacturer’s recommendations. All reactions were performed in triplicate and at least three independent experiments.

HepG2 cells (DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) were cultured in RPMI 1640 GlutaMAX™-I supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. Hep-3B cells (DSMZ, Braunschweig, Germany) and Huh7 cells (JCRB Cell Bank, Tokyo, Japan) were cultured in DMEM supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin.
Cells were detached with TrypLE Express W/Phenol red (Life Technologies, Darmstadt, Germany). Media and additives were obtained from Gibco (Life Technologies). Cells were cultured under standard conditions at 37 °C in a humidified atmosphere supplemented with 5% CO₂.
For transfection experiments, 1.5 × 10⁵ Huh7 and Hep-3B cells, respectively, and 2 × 10⁵ HepG2 cells were plated per well of a 12-well plate (Nunc, Langenselbold, Germany) and grown for 24 h to reach approximately 80% confluence. Lipofectamine 2000 (Invitrogen, Karlsruhe, Germany) was used to transfect the cells. Per well, 4 µL Lipofectamine, 1.6 µg plasmid DNA and 3 ng pRL-CMV Renilla luciferase control vector (Promega, Mannheim, Germany) were applied. The cells were harvested and luciferase activity was measured as described previously [24 (link)].

Transfection of the NG108-15 cells was carried out using lipofectamine (Invitrogen) following the manufacturer's protocol. For each well (48-well tissue culture plates; 80% confluency), 0.5 μg Ca_V3.2 luciferase reporter plasmid, 0.0125 μg pRL-TK (Promega) and 0.5 μl lipofectamine were mixed with 25 μl serum-free medium. The mixture was incubated for 20 min at room temperature and then added to the appropriate wells. Cells were grown in serum-free culture medium at 37 °C and 5% CO₂. After 16 h, the serum-free medium was replaced by serum-containing medium and the cells were used for experiments 36 h after transfection.
Renilla luciferase was used to normalize the transfection efficiency data, and a Dual Luciferase Reporter Assay System was used according to the manufacturer's specifications (Promega). Renilla and firefly luciferase activities were determined using the Glomax Luminometer (Promega). The results are given as firefly/Renilla relative light units if not indicated otherwise.

The CRIM1 fragments containing the binding site for miR-199b-3p were cloned into the luciferase reporter vector (Promega, WI, USA) (CRIM1 WT). Mutation (MUT) sites were also designed (CRIM1 MUT). Cells were transferred into 24-well plates at 3 × 10⁴ cells per well. After 24 h, the cells were transiently co-transfected with 0.1 μg/well of the luciferase reporter vector (CRIM1 WT or CRIM1 MUT) and 50 nM miR-199b-3p mimic/NC mimic into SW480-CTxR/HCT116-CTxR cells using Lipofectamine® 2000 reagent for 48 h, the cells were collected to test the activities of firefly and Renilla luciferase by the Dual-Luciferase Assay System (Promega, WI, USA). Relative luciferase activity was expressed as the ratio of Renilla/firefly.