Fig 2: Biochemical characterization of rat liver subcellular fractions and Pgp localization in endolysosomal-enriched fractions. Rat liver subcellular fractionation was performed by low speed centrifugation and subsequent density gradient ultracentrifugation. Recovery and purity of the cell organelles from collected gradient fractions 1–10 (top to bottom) were analyzed by separation of equal protein amounts (5 µg) of each fraction and whole-cell homogenate (H) and post-nuclear supernatant (PNS) by SDS-PAGE and immunoblotting. Homogenate and PNS data were similar; thus, PNS is shown here for only some proteins. Endolysosomes were detected using the LAMP-2 and Rab7 marker proteins and the luminal lysosomal protease cathepsin D (CatD). EEA1 was used as a marker for early endosomes. The purity of the endolysosomal fractions was assessed by organelle markers for mitochondria (VDAC), canalicular plasma membrane (ABCB4), and ER (calnexin). (A) Representative Western blots showing the distribution of the different organelle protein markers and Pgp in the gradient fractions. Some fractions (mainly 2 and 3) contained low protein amounts and thus could not be characterized for the presence of all marker proteins for each individual gradient. Therefore, marker quantification in the gradient fractions was repeatedly performed for different fractionations. (B) Quantification of the protein band intensities for the marker proteins in the different gradient fractions. Immunoblotting revealed recovery of endolysosomal vesicles from the top of the gradient (fractions 1–4) and co-fractionation of Pgp in the enriched endolysosomal fractions (1–4). Fraction 1 was essentially free of canalicular plasma membrane contamination and mitochondria but contains remnants of ER. Mitochondria localized in fractions 7–10 and were clearly separated from endolysosomal markers. The ER marker calnexin was present in each fraction, with the lowest amount in endolysosomal fraction 1. Values are expressed as the percentage of total marker protein levels detected in all fractions combined (set to 100%). Data are presented as means ± SEM; the number of quantified Western blots (n) is shown below the graphs.

Fig 3: Effect of the protein expression of FXR (A, E), CYP7A1 (B, F), BSEP (C, G) and MDR2 (D, H) in mice after PM-L and PM-H treatment for 8 weeks. Quantification of the protein expression was performed by densitometric analysis of the blots following normalization to GAPDH expression. (mean ± SEM, n = 3, *p < 0.05, **p < 0.01).

Fig 4: Transcellular transport of TKIs in the ABCB4 and Mock cells. (a) Bidirectional transport of gefitinib. ABCB4 and Mock cells were incubated with 0.5 µM gefitinib for 60, 120 or 180 min. Data are represented as means ± S.D. for three experiments. Gefitinib concentration in the samples was determined with LC-MS/MS. (b) Bidirectional transport of imatinib. ABCB4 and Mock cells were incubated with 0.5 µM imatinib for 60, 120 or 180 min. Imatinib concentration in the samples was determined with LC-MS/MS. Data are represented as means ± S.D. from three independent experiments.

Fig 5: Efflux ratios for prototypic ABCB1 substrates. (a) Efflux ratios for digoxin and talinolol (1 µM, traced with 0.17 µCi/mL 3H-labelled probe) in the MDCKII parental and in the Abcb1KO cells. The dashed line indicates an efflux ratio of 2. Data are presented as mean ± standard deviation for 3 experiments each performed in triplicate. (b) Functional characterization of ABCB1, Mock and ABCB4 cells using prototypical ABCB1 substrates (1 µM, traced with 0.17 µCi/mL 3H-labelled probe). The dashed line indicates an efflux ratio of 2. Data are presented as mean ± standard deviation for 3 experiments each performed in triplicate.