Fig 2: Cholesterol enters HepG2 and differentiated Caco-2 cells by NPC1L1-independent manner mainly.A, (a) Transient overexpression of NPC1L1 in the plasma membranes examined by Western blotting analysis. Upper, QuickBlue staining; lower, immunostaining for NPC1L1. (b) ABCG5 (upper) and ABCG8 (lower) protein expressions in HepG2 cells. Insets, these two images were adjusted to increase visibility of the bands. B, Medium-to-cell cholesterol transit in HepG2 cells. Transfection and ezetimibe treatment were performed as indicated as shown in the bottom. Medium-to-cell 3H-cholesterol transit efficiency (%) was estimated as described in the “Materials and Methods”. Each plot shows an individual assay result. Bars indicate means. Alphabetical differences among the groups (in parentheses) indicate significant difference between the groups (p < 0.05) using Tukey's Honestly Significant Difference test. C, Cholesterol (upper) and protein (lower) abundance in HepG2 cells. No significant difference was observed among the groups using Tukey's Honestly Significant Difference test. Bars show mean ± SEM (n = 4). D, (a) An illustration of Caco-2 cell culture system used in this study. Caco-2 cells were grown on filter membranes to allow the development to an enterocyte-like phenotype (see text for the detailed methods). The supernatants in culture inserts and wells were designated as apical and basolateral media, respectively. Lipid micelles were added to the apical medium. (b) Absorptive epithelial cell morphology of differentiated Caco-2 cell monolayers with a cylinder-like cell shape, the development of microvilli, and the distal localization of nuclei demonstrated by an electron microscopic analysis. (c) NPC1L1 was localized to the apical membrane in differentiated Caco-2 cells. Confocal microscopic analysis showed that NPC1L1 colored in green was localized to the plasma membrane (the upper image), especially in the brush border area (the lower image). 7-amino-actinomycin D was used for counterstaining of nuclei (red). E, Ezetimibe had little effect on medium-to-cell 3H-cholesterol transit in differentiated Caco-2 cells. Open circles, vehicle (1% ethanol); closed circles, 50 µmol/l ezetimibe. Data were shown as mean ± SEM of triplicate assays. F, In contrast to human duodenum (a) and murine jejunum (b), the gene expressions of ABCG5 and ABCG8 were absent in differentiated Caco-2 cells (c). Gene expression levels of five major membrane sterol transporters were analyzed by quantitative RT-PCR. An RNA sample of human duodenum was assayed in triplicate. Murine jejunal RNA samples were obtained from three C57BL/6J mice and assayed in duplicate. Three separate total RNA samples were obtained from independent wells of differentiated Caco-2 cells and assayed in duplicate. Data were shown as mean ± SEM of analytical triplicate (a) or biological triplicate (b, c) assays.