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Supplemental figure 1.

Comparison to DMSO and Adult bovine serum mediated growth arrest Historically, fetal serum has been used in cell cultures because of the presence of high levels of growth stimulating factors. We wanted to determine if some of the same effects seen in HS (adult human serum) could be achieved by switching FBS to ABS (adult bovine serum). Also, HuH-7 or HuH-7-derived cells become contact inhibited by DMSO, as reported previously (4). We therefore characterized Huh7.5 cells grown in either FBS media further supplemented with DMSO (1%) or in media supplemented with adult bovine serum (ABS, 2%).

Both ABS and DMSO supplementation resulted in growth arrest and changes in cellular morphology, however these changes were less pronounced than in HS; for example the increase in cells size that was observed under HS conditions was not observed in DMSO or ABS containing medium. The degree of differentiation was further examined by quantitative RT-PCR for differentiation markers, cell-cell contact components and key lipid regulators, and compared with cells cultured in HS supplemented media (supplemental figure 1). Of the hepatocyte specific markers, only albumin was increased in DMSO, but not in ABS, and did not reach the level found in HS media. However, DMSO supplementation also resulted in a decrease in LDL-R transcripts. (suppl. figure 1A). Of the cell-cell contact markers, only occludin was increased by supplementation with ABS, and again not to levels found in HS media (suppl. figure 1B). The levels of key lipid regulators were not affected by culturing in DMSO or ABS, with the exception that LXRα was significantly decreased in DMSO cultured cells (suppl. figure 1C). In line with this, the increase in lipid droplet content that was observed in HS was not seen in cells cultured in either DMSO or in ABS (not shown).

Summarizing, although both DMSO and ABS induce growth arrest and some of the same morphological changes as seen in HS, and results in expression of some differentiation markers, neither of them induces all changes, nor at similar levels that HS supplementation does.

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