LDL Uptake Assay

LDL uptake was measured using a BODIPY^® FL kit. HepG2 cells were seeded into 48-well plates at 4 × 10⁴ cells per well in growth medium and allowed to adhere to the plate for 24 h. After serum deprivation for 16 h, the cells were treated with or without 0.01–10 μM βE2 for 4 h in the presence of rhPCSK9 (25 μg/mL). For the assay, the cells were pretreated with 1 μM G15 for 15 min prior to the addition of βE2 to block GPER action. BODIPY^TM FL LDL (6 μg/ml) was added for 3 h, and then the cells were then washed with PBS. Isopropyl alcohol was added to measure the uptake of BODIPY^TM FL LDL. The plates were read with a SpectraMax M5 reader (Molecular Devices, Sunnyvale, USA).