Cycloheximide study

HEK293T cells were maintained in DMEM containing GlutaMAX (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Thermo Fisher Scientific) at 37°C in a 5% CO₂ atmosphere. Cells were plated in a 6-well plate (200 000 cells per well). 24 hr later, cells were transiently transfected with the indicated plasmids using Fugene Transfection Reagent (Promega) according to the manufacturer’s instructions. 24 hr later, the culture medium was aspirated and replaced with fresh culture medium containing 100 μg/mL cycloheximide (Sigma-Aldrich - Ready-Made solution in DMSO - 100 mg/mL) or the equivalent volume of DMSO for the indicated times.