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Figure 1. Phenotypic characterisation of HepG2-EV, -UGT1A1 and -UGT2B7 cell clones. (A) Representative phase contrast microscopy images of HepG2 cell clones and HepG2 parental cells cultured as exponentially growing monolayers. Scale bar: 100 µm; (B) PDT of exponentially growing cultures of HepG2-UGT/-EV clones and parental HepG2 cells. No statistically significant differences were identified using one-way ANOVA and Tukey’s multiple comparisons test with three biological replicates; (C) Relative UGT1A1 (left) and UGT2B7 (right) mRNA expression levels in HepG2-UGT/-EV cells determined by qRT-PCR. Statistical analysis was conducted using one-way ANOVA followed by Tukey’s test (comparing to parental HepG2 cells) with three biological replicates; (D) Relative ABCB1, ABCC2, and ABCG2 mRNA expression levels in HepG2-UGT/-EV cells as determined by qRT-PCR. Statistical analysis was conducted using one-way ANOVA followed by Tukey’s test (compared to parental HepG2 cells, which served as a reference) with three biological replicates; (E) Protein expression of UGT1A1 and UGT2B7 (visualised by red fluorescence) in HepG2-UGT/-EV clones and parental HepG2 cells as determined by immunofluorescence staining with DAPI counterstaining to visualize cell nuclei (blue fluorescence); representative images are shown. Scale bar: 100 µm; (F) Quantification of UGT1A1 (left) and UGT2B7 (right) immunofluorescence in HepG2-UGT/-EV cells, expressed as mean fluorescence intensity (MFI; red channel) per cell; (G) Specific UGT activity in microsomal fractions prepared from HepG2-EV, -UGT1A1 and -UGT2B7 cell clones as determined by the fluorometric UGT Activity Assay/Ligand Screening Kit. Statistically significant differences were identified using a two-tailed unpaired t-test with four independent replicates. Significance levels are indicated as follows: ns = not significant (P > 0.05), ^**P < 0.01, ^***P < 0.001 and ^****P < 0.0001. PDT: Population doubling times; ANOVA: analysis of variance; mRNA: messenger RNA; qRT-PCR: quantitative real-time polymerase chain reaction; DAPI: 4′,6-diamidino-2-phenylindole; MFI: mean red fluorescence intensity; UGT: uridine diphosphate glucuronosyltransferase.