3A,B), which was similar to the levels of expression noted in cultured primary hepatocytes. We also found that e-cadherin is up-regulated approximately 6-fold in cells cultured in HS media (Fig. 3C). LDL-R, claudin-1, and occludin have also been recognized as factors involved Palbociclib concentration in HCV entry. To investigate alterations in some of the other factors involved in entry of HCV, we also determined mRNA levels of CD81, scavenger receptor class BI (SR-B1), and Niemann-Pick C1-like 1 (NPC1L1). No changes were observed in mRNA levels of any of these entry factors as a result of culturing in HS-supplemented media (Fig. 3D-F). The cytoplasm of cells in HS media had a prominent granular appearance.

To determine whether this change in morphology was the result of alterations in the amount of lipid droplets, cells were stained with Bodipy 493/503, a lipophilic fluorophore with a high affinity for lipid droplets. We found that Bodipy fluorescence intensity was approximately 4× higher in Huh7.5 cells in HS media than in Huh7.5 cells cultured in FBS (Fig. 4A-C). We next investigated the expression of three key lipid regulators: liver X receptor α (LXR-α) and peroxisome proliferator-activated receptors (PPAR-α and PPAR-γ). LXR-α is highly expressed in liver, is activated by cholesterol metabolites, and

regulates genes involved in cholesterol processing and secretion.[11] Consistent with increased lipid droplet contents, we found that LXR-α find more expression is highly increased in cells cultured in HS,

compared to FBS (Fig. 4D). Transcription of PPAR-α as well as PPAR-γ was up-regulated significantly in cells cultured in HS, compared to FBS (Fig. 4E,F). PPAR-α is highly expressed in liver and regulates mitochondrial function, fatty acid uptake, beta-oxidation, and TG metabolism, as well as lipoprotein assembly.[11] PPAR-γ also regulates genes involved in lipid metabolism and is activated by an array of ligands, including unsaturated fatty acids.[11] Importantly, we wanted to determine whether Huh7.5 cells cultured in HS media regain some of the complex functionality of primary hepatocytes that is considered lost in Methisazone FBS-cultured Huh7.5 cells. The ability to secrete nascent VLDL particles is one example of such a complex process[12] because it depends on the integration of biogenesis, modification, and transportation processes. In line with previous observations,[7, 13] VLDL secretion is virtually absent in Huh7.5 cells that are grown in FBS-supplemented serum (Fig. 5). In cells cultured in HS media, VLDL secretion is gradually restored when cells are cultured in HS: After 5 days, minor changes can be noted on the triacylglyceride- and cholesterol-based lipoprotein profiles (Fig. 5A,B), and by 14 days, a prominent VLDL peak appears in HS-cultured cells. Also, the LDL peak increases in size and elutes earlier, indicating larger particles.