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. 2020 Jan 14;9:e50135. doi: 10.7554/eLife.50135

Figure 5. CYP3A4 expression levels and induction in Duodenum Intestine-Chip and Caco-2 Intestine-Chip.

Figure 5.

(a) Average gene expression levels of CYP3A4 ± s.e.m (error bars) in Caco-2 Intestine-Chip, Duodenum Intestine-Chip assessed at day 8 of their culture and human duodenum (three independent biological specimens). All values are shown relative to the adult duodenal tissue expressed as 1, one-way ANOVA, ****p<0.0001, **p<0.01. EPCAM expression was used as normalizing control. (b) Protein analysis of CYP3A4 in Caco-2 and Duodenum Intestine-Chip assessed at day eight using western blot. (c) The CYP3A4 induction in Duodenum Intestine-Chip and Caco-2 Intestine-Chip treated at day 6 with solvent (DMSO), 20 μM rifampicin (RIF) or 100 nM 1,25-dihidroxyvitamin D3 (VD3) for 48 hr. The gene expression levels (top) of CYP3A4 were examined by real-time PCR analysis at day 8. On the y-axis, the gene expression levels in the DMSO-treated chips were taken as 1.0. All data are represented as means ± s.e.m. Two-way ANOVA, ****p<0.0001 (compared with DMSO-treated cells). The corresponding CYP3A4 protein expression levels (bottom) were measured at day 8 by western blotting analysis. (d) CYP3A4 enzyme activity was determined by monitoring the formation of 6β-hydroxytestosterone in the medium of Duodenum Intestine-Chip and Caco-2 Intestine-Chip, as measured by LC-MS. For induction studies, 20 µM RIF or 100 nM VD3 was added 48 hr before measurement. Data are expressed as mean ± s.e.m of three independent experiments each involving Duodenum Intestine-Chip established from organoid-derived cells of a different donor and Caco-2 Intestine-Chip. At least three different chips were used per condition. Two-way ANOVA, ****p<0.0001 (e) Gene expression analysis of the receptors PXR and VDR in Duodenum Intestine-Chip and Caco-2 Intestine-Chip examined at day 8 by real-time PCR analysis and compared to their expression in adult duodenal tissue (Duodenum). On the y-axis, the gene expression levels in adult tissue were taken as 1.0. All data are represented as means ± s.e.m. Two-way ANOVA, ***p<0.001, **p<0.01, *p<0.05.