Fig. 2.

CE metabolism in Huh7 cells. A–C: HPTLC autoradiograms for time course for Huh7 cell metabolism of [¹⁴C] CE taken up from HDL, CERM and LDL. Cells were pulsed for 2 h with the respective [¹⁴C] CE radiolabeled lipoprotein at 14.3 nmol CE/mL, and chased for 0, 2, 4 or 6 h prior to harvest of cells for analysis. Total time (pulse + chase) is indicated, and triplicate samples are shown for each time point. The lower panels, labeled CE, show the primuline stain of the endogenous CE present in the samples. D–F: Kinetic curves for cellular CE hydrolysis and cellular FC and oxysterol formation, respectively. Three oxysterol bands were detected, and the sum of the volume of these was plotted as the rate of formation of oxysterols. Amounts at each time point were calculated by phosphor image analysis of the autoradiograms, using ImageQuant software. The data are the average of two independent experiments, each done in triplicate. Significant differences (p < 0.05) at each time point are indicated by symbols: α, HDL vs CERM;β, HDL vs LDL;χ, CERM vs LDL. Kinetic constants were determined for CE and FC according to exponential decay and rise to maximum, respectively. The rate of oxysterol appearance was calculated from a first order linear polynomial. Rate constant values are given in Table 1.