Acetyl-CoA and CoA analysis by HPLC

WT and ACAT1 KO cells were plated at a density of 5×10⁶ cells per 10-cm culture dish and cultured in DMEM (10%FBS) for one day. Cells were washed with PBS, detached from the dish using CellStripper Solution. Cell pellet was collected by centrifugation, washed with PBS twice, and resuspended in 100 μl of 5% 5-Sulfosalicylic acid (Sigma) solution. To permeabilize the cells, samples were frozen in liquid nitrogen and thawed on ice. This freeze–thaw cycle was repeated twice. After centrifugation, the supernatant was filtered using Ultrafree-MC LH Centrifugal Filter (Millipore, Billerica, Massachusetts, UFC30LH25). Then, the samples were transferred into SUN-SRi Glass Microsampling Vials (Thermo Fisher Scientific, 14–823-359) with SUN-SRi 11mm Snap Caps (Thermo Fisher Scientific, 14–823-379), and 80 μl of each sample was separated using an Agilent 1100 HPLC (Agilent Technologies, Santa Clara, California) equipped with a reverse phase column, Luna 3 μm C18(²) 100 Å, 50 ×4.6mm, 3 μm (Phenomenex, Los Angeles, California). The detailed protocol was described previously (⁴⁴).