Mammalian cell growth and mRNA transfection

HEK293T cells were cultured and transferred into 24-well plates 12–16 h before transfection, as described³⁹. The transfection was performed using Unifectin-56 (Unifect Group, Russia). The standard protocol was modified to obtain maximal mRNA transfection efficiency according to⁴¹. Amicoumacin A ethanol stock was diluted with water to obtain 100x solutions, as indicated, and was added to the medium right before addition of the transfection complexes. All manipulations were performed in such a way to minimize time of holding the cells out of CO₂ box and to avoid cooling the plate. Two hours after the transfection, cells were harvested, and luciferase activities were analyzed with the Dual Luciferase Assay kit (Promega). All the transfections were repeated at least three times in different cell passages.