Figure 5
Characterization of monolayer model for ADME assays. (a) Whole mount immunostaining of differentiated monolayer culture which stained with E-cadherin (green), ZO-1 (red), F-actin (white) with DAPI (blue). Scale bar = 100 µm (b) Whole mount immunostaining of differentiated monolayer culture stained with P-gp (red), phospho-Ezrin (green), or villin (green) together with DAPI (blue). (c) Production of 1-hydroxymidazolam with and without 1 mM ABT in differentiated monolayer with 48 h exposure of 100 nM VD3. (d) Transporter assay using P-gp substrate 10 µM digoxin which was exposed from apical or basal side of monolayer culture with and without 5 µM Zosuquidar (ZSQ). (e) CYP1A activity and CYP3A activity were analyzed by P450-Glo Assays. Expression of Cyp1a1, Cyp3a9, Abcb1a, Abcb1b, Abcc2 were evaluated by RT-PCR. Data represents mean ± s.d. (n = 3, biological replicates). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, Dunnett’s test compared with the control for CYP1A induction assay, Student t test for digoxin transport assay and CYP3A induction assay. Papp apparent permeability coefficient, ADME absorption, distribution, metabolism, and excretion, ABT 1-aminobenzotriazole, ZSQ zosuquidar, AB apical to basal, BA basal to apical, βNF beta-naphthoflavone, 3MC 3-methylcholanthrene, VD3 1alpha, 25-dihydroxyvitamin D3.
