Preparation and Transfection of Mouse Embryonic Fibroblasts

Mouse embryonic fibroblasts (MEFs) were cultured as previously described^{72 (link)}, using Dulbecco’s modified eagle medium (DMEM, Thermofischer Scientific, 41965-039) supplemented with 10% v/v FBS (Thermofischer Scientific, 10270-106), 1% v/v penicillin-streptomycin (Thermofischer Scientific, 10378-016), and 1.5% v/v HEPES 1M (Sigma Aldrich, H0887). Cell cultures were routinely checked for mycoplasma. CO₂-independent media was prepared by using CO₂-independent DMEM (Thermofischer Scientific, 18045054) supplemented with 10% v/v FBS, 1% v/v penicillin-streptomycin, 1.5% v/v HEPES 1M, and 2% v/v L-Glutamine (Thermofischer Scientific, 25030-024). Media for AFM experiments was supplemented with Rutin (ThermoFischer Scientific, 132391000) 10 mg/l right before the experiment. Importazole (Sigma Aldrich) was used at 40 μM concentration for 1 h^{56 (link)}. Cells were transfected the day before the experiment using Neon transfection device (ThermoFischer Scientific) according to manufacturer’s instructions. Cells were seeded ~4 h before the experiment.