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Fig. 3 Increased lipid deposition was found in FD Huh7 cells. Huh7 cells were cultivated in FD medium and examined for cell viability and lipid content. a Huh7 cells cultivated in CTL (0.004 g/L folic acid) or folate-deficient (FD) medium were analyzed with microbiology assay for folate content. A more than 90% depletion of intracellular folate content was established at 10 day-post-seeding (dps) for the cells growing in FD medium. Presented are data collected from 3 and 4 independent experiments at 5 dps, and 8 and 10 dps, respectively. b Huh7 cells were cultivated in CTL or FD medium for 7 days (dps) before adding FA or 5-CHO-THF for rescue. Cell viability was examined on 8 dps. The decreased cell viability due to FD was successfully rescued by the presence of FA or 5-CHO-THF. Data were collected from at least 6 independent experiments. c Cells were stained with Nile red and analyzed for lipid content with direct visual inspection with fluorescence microscopy (left) and quantified by flow cytometry (right). Scale bars = 40 µm. Data were collected from at least 7 independent experiments. d-e The cholesterol (d) and triglyceride (e) levels of Huh7 cells at 8 dps was measured with colorimetric/fluorometric Assay. Data were collected from at least 6 independent experiments. f-g Cells cultivated in FD medium with/without FA or 5-CHO-THF supplementation were examined for ALT (f) and AST (g) content. The increase of both ALT and AST was effectively alleviated by supplementing with FA or 5-CHO-THF. Presented are the averaged results of at least 8 independent trials. CTL, control (cells without FD); FD, folate deficiency. Statistical data are shown in mean ± SEM. * p<0.05, **, p <0.01; ***, p<0.001, ****, p<0.0001