LDL uptake assay

Blood was drawn from healthy volunteer, and LDL was prepared by ultracentrifugation as previously described [25]. LDL was then labeled with DyLight-488 (Thermo Fisher Scientific) following manufacturer’s protocol, and LDL uptake was measured using DyLight-488-labeled LDL as previously described [26]. In brief, HepG2 and Huh7 cells were cultured for 16 hours prior to adding LDL in sterol-depleted medium [(DMEM supplemented with 10% bovine lipid-deficient serum (LPDS), 5 μg/mL simvastatin (Selleck), and 100 μM mevalonic acid (Sigma Aldrich)] as previously described [5]. Cells were then incubated with 5 μg/mL DyLight-488-labeled LDL in DMEM containing 0.5% BSA for 3 hours at 37°C or 4°C. In these experiments, 100 μg/mL non-labeled LDL was used to correct for non-specific LDL binding. After incubation, cells were washed twice by ice-cold PBS supplemented with 0.5% BSA and then lysed in RIPA buffer. Specific LDL uptake was calculated as the fluorescent intensity differences between 37°C and 4°C, after subtracting non-specific association/binding. LDL uptake was determined by quantification of the fluorescence signal using a multi-mode fluorescent microplate reader (Cytation 5, Biotek), and corrected for the protein contents in the same lysates as determined with the BCA assay. To visualize LDL uptake, cells were cultured on coverslips and treated as above mentioned, then fixed with 4% paraformaldehyde, and mounted with Vectorshield containing DAPI (Vector Laboratories). Prepared slides were visualized with Cytation 5 using slide imaging mode with a 40x objective lens.