Effect of hypertonic medium on internalization of WT-PCSK9-Red. Conditioned medium from HepG2 cells transiently transfected with WT-PCSK9-Red plasmid was made hypertonic by addition of NaCl which increased the final concentration of NaCl by 150 mmol/l. The hypertonic, conditioned medium was added to untransfected HepG2 cells for 3 h incubations at 37°C or at 4°C. Isotonic, conditioned medium is conditioned medium without addition of NaCl. Amounts of cell-associated WT-PCSK9-Red were determined by flow cytometry. Control is medium without WT-PCSK9-Red. Results represent mean (± SD) of three experiments (** p < 0.01, *** p < 0.001).