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Additional Supporting Information may be found in the online version of this article.

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HEP_25955_sm_SuppFig1.tif2558KSupporting Information Figure 1. Illustration of automated membrane identification and fluorescence intensity profile extraction in confocal images: In fluorescence images of Ntcp (red) and Na+K+-ATPase (green) (A), basolateral membranes are identified by foreground-background detection. White foreground regions (B) are skeletonized (C). At every pixel of the skeleton, fluorescence intensity profiles are extracted perpendicular to the skeleton segments. Red line in figure D exemplarily demonstrates profile length (8μm) and optimal intensity profile orientation. Intensity profiles are extracted both for the “structural” (Na+K+-ATPase) and “functional” marker (Ntcp). Na+K+-ATPase profiles undergo selection by empirically identified criteria and ranking according to quality parameters. Intensity profiles from white membrane segments were accepted for statistical profile evaluation after profile selection (D).
HEP_25955_sm_SuppFig2.tif1763KSupporting Information Figure 2. Supporting figure 2: TLCS, TLC and TCDC induce retrieval of Ntcp from the sinusoidal membrane in flow cytometry: HepG2 cells stably expressing FLAG-Ntcp-EGFP were cultured at subconfluence, treated with 100μM of different bile salts (1h at 37°C) and analyzed by flow cytometry. Fluorescence intensity distributions are shown in cumulative histograms. Cells showed significant reduction of FLAG-associated fluorescence after pre-incubation with 100μM TLCS, TLC or TCDC, compared to TC control (A), whereas green fluorescence (EGFP) was not affected (B). General cell morphology was not affected by bile salt stimulation, as shown by representative images from TC or TLC treated cells (C, D).
HEP_25955_sm_SuppFig3.tif4291KSupporting Information Figure 3. Supporting figure 3: TCDC, but not TC, induces Ntcp retrieval from the plasma membrane in perfused rat liver: Perfusion with TCDC (C,D), but not TC (A,B), led to an internalization of Ntcp from the plasma membrane into the cytoplasm, while Na+K+-ATPase immunostaining remained unchanged under both conditions (E-H). Bar=10μm.
HEP_25955_sm_SuppFig4.tif4560KSupporting Information Figure 4. Supporting figure 4: TCDC, but not TC perfusion, significantly increases the amount of Ntcp colocalizing with the early endosome marker EEA1: Co-immunofluorescence staining of Ntcp and EEA1 before and after perfusion with 100 μmol/l TC (A,C) or TCDC (E,G), respectively. Greyscale images show colocalizing pixels of the red and green channel (B,D,F,H). The colocalization pattern of Ntcp and EEA1 indicates an EEA1 distribution in a submembraneous compartment (B,F). Bar=10μm. Colocalizing pixels were quantified by calculation of the weighted colocalization coefficient using LSM510 Meta 4.2 Software (Zeiss, Jena, Germany). In images from tissue sections of TCDC perfused rat liver colocalization was significantly increased. ** indicate statistically significant differences (p<0.005)(I).
HEP_25955_sm_SuppFig5.tif2887KSupporting Information Figure 5. Supporting figure 5: Subcellular Bsep distribution is not affected by TC or TCDC perfusion: Cryosections from perfused rat livers were prepared for confocal microscopy by immunohistochemistry and analyzed by automated image processing. The numerical descriptor y was introduced to assess transport protein translocation from the canalicular membrane into the cells. It describes the ratio between fluorescence intensities in the center of the canaliculus (zone 1) and intracellular fluorescence intensities (zone 2a, 2b, 3a and 3b) (A). Descriptor z was developed for statistical analysis of the structural marker protein (Zo-1). It encompasses the ratio between the peak fluorescence intensities (zone 2a and 2b) and intracellular fluorescence intensities (zone 3a, 3b, 4a and 4b). Descriptor z (E) values were calculated for each individual Zo-1 intensity profile. As another quality criterion, data sets were only accepted if Zo-1 distribution was unchanged (G, I). Accordingly, descriptor y (E) values were calculated for each individual Bsep intensity profile. The Wilcoxon rank sum test was applied to compare data sets. Neither TC (F) nor TCDC (H) perfusion resulted in a significant shift in descriptor y values, indicating that subcellular Bsep distribution remains unchanged under both conditions (A-D, bar=10μm).
HEP_25955_sm_SuppFig6.tif2232KSupporting Information Figure 6. Supporting figure 6: Comparative analysis of the effects of different bile salts on TC* recovery in effluent, biliary secretion and bile flow: Livers were perfused with 100μmol/l of TC throughout the experiment. Different bile salts were perfused prior to a pulse of 3[H]-TC (grey box)(see methods section for experimental details). Diagrams represent TC* recovery from the effluent (A, D and G), TC* secretion in the bile (B, E and H) and bile flow (C, F and I).
HEP_25955_sm_SuppFig7.tif169KSupporting Information Figure 7. Supporting figure 7: Pre-perfusion with TLC exceeds the effect of TCDC in increasing TC* recovery from effluent: TLC at a concentration of 40μmol/l significantly reduces net sinusoidal uptake at a comparable degree as 100 μmol/l of TCDC. Higher concentrations of TLC led to complete stop of bile flow (A). Stimulation with different bile salts (TC, TCDC and TLC) affects the amount of TC* secreted into bile, but the time profile of biliary secretion is almost unaffected (B).
HEP_25955_sm_SuppFig8.tif215KSupporting Information Figure 8. Supporting figure 8: Inhibition of bile salt uptake by TCDC is non-competitive: Livers were perfused with 100μmol/l of TC throughout the experiment. 100μmol/l of TC were perfused prior to a pulse of 3[H]-TC (A). In order to rule out a competitive mechanism of TCDC-induced inhibition of TC secretion, the basic experimental setup was modified by replacement of TC and 3[H]-TC by TCDC and 3[H]-TCDC. The amount of TCDC* recovered from the perfusate was four times higher (B) as compared to TC perfusion (A), while bile flow was markedly reduced (C versus D).
HEP_25955_sm_SuppFig9.tif2910KSupporting Information Figure 9. Supporting figure 9: Comparative analysis of the effects of different inhibitors on TC* recovery in effluent, biliary secretion and bile flow: Livers were perfused with 100μmol/l of TC throughout the experiment. Inhibitors were added 20min before stimulation with 100μmol/l of TC or TCDC (see Fig. 2 for details), followed by a 5min pulse of 3[H]-TC (grey box). Diagrams represent TC* recovery from the effluent (A, D and G), TC* secretion in bile (B, E and H) and bile flow (C, F and I).
HEP_25955_sm_SuppFig10.tif2326KSupporting Information Figure 10. Supporting figure 10: Comparative analysis of the effects of different inhibitors on TC* recovery in effluent, biliary secretion and bile flow: Livers were perfused with 100μmol/l of TC throughout the experiment. Inhibitors were added 20min before stimulation with 100μmol/l of TC or TCDC (see Fig. 2 for details), followed by a 5min pulse of 3[H]-TC (grey box). Diagrams represent TC* recovery from the effluent (A, D and G), TC* secretion in bile (B, E and H) and bile flow (C, F and I).
HEP_25955_sm_SuppInfo.rtf197KSupporting Information

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