Genjet

HEK293 cells were cultured in Dulbecco’s modified eagle medium (DMEM) high glucose (Gibco), supplemented with 10% fetal bovine serum, 1.5% HEPES, 1% minimal essential medium non-essential amino acids (MEM NEAA), and 1% sodium pyruvate, in a humidified incubator at 37°C and 5% CO₂. Twenty-four hours prior to transfection, HEK293 cells were seeded into six-well plates (Cellstar^® 6-well Cell culture multiwell plate, Greiner Bio-One) with 5 x 10⁵ cells per well in 5 ml cell culture medium, and incubated at 37°C and 5% CO₂. Transfection was performed using GenJet (Signagen) according to the manufacturer’s protocol using 2 µg DNA in 100 µl cell culture medium per well without supplements and 6 µl GenJet in 100 µl cell culture medium per well without supplements. Cells were incubated with the transfection reagents for approximately 20 h at 37°C and 5% CO₂. Subsequently, transfection reagents were removed from the cells and exchanged for cell culture medium with supplements. The cells were incubated at 37°C and 5% CO₂ for 24 h before seeding for testing.

Assays were performed as described [9 (link)] with the following modifications: 5 × 10⁴ Hela PKR^kd cells per well were seeded in 24-well plates 16 hours prior to the experiment. Cells were transfected with 200 ng of the indicated PKR expression vector, the indicated amount of each K3L expression vector, and 50 ng of pGL3 firefly luciferase expression vector (Promega) using GenJet (Signagen) at a DNA to GenJet ratio of 1:2 following the manufacturer’s protocol. Empty pSG5 vector was used to maintain the DNA concentration at 200 or 100 ng where appropriate. 48 hours post-transfection cells were lysed with mammalian lysis buffer (GE Healthcare), then luciferin (Promega) was added following the manufacturer’s recommendations. Luciferase activity was measured using a GloMax luminometer (Promega). Experiments were conducted in triplicate in three independent experiments.