Efect of hypertonic, conditioned medium on internalization of LDL and on the amount of cell surface LDLR. HepG2 cells were transiently transfected with empty plasmid, WT-PCSK9-His plasmid or D374Y-PCSK9-His plasmid. Conditioned media from transfected cells were made hypertonic by addition of NaCl which increased the final concentration of NaCl by 150 mmol/l. The hypertonic medium was added to untransfected HepG2 cells for a 3 h incubation at 37°C. Isotonic, conditioned medium which had not been added NaCl was used as control. A) To measure internalization of LDL the cells were added fluorescently labelled DiD-LDL (10 μg/ml) and incubated for 2 h at 37°C. B) To measure the amounts of cell surface LDLR, the cells were labelled with anti-LDLR IgG-C7 antibody and counter-stained with Alexa Fluor® 488 goat anti-mouse IgG. Cell fluorescence was quantified by flow cytometry. Results represent mean (± SD) of three experiments (* p < 0.05, ** p < 0.01, *** p < 0.001).