Fig 2: PPL expression after recovery from dietary CA-induced cholestasis. (A) Dietary administration schedule. Seven weeks-old male wild-type mice fed NMF diet were separated in two groups. One group (Recovery) was given the 1% CA-containing NMF diet for 1 week to induce cholestasis; the diet was then replaced by the control NMF diet to resolve the cholestatic situations. The other group (Control) was given the control NMF diet throughout the experiment. Mice were sacrificed in each group at the time points indicated by arrows. (B) The levels of the serum markers for cholestasis (T-Bil and T-BA; left panel) and hepatocellular injury (ALT and AST; right panel) at each time point in control and recovery groups. Values are shown by the symbols and colors as indicated in inlets. Averages from 5 mice are expressed with standard errors. (C) Hepatic PPL expression examined by immunohistochemical and western blot analyses. Representative images resulting from immunohistochemical analysis are shown for each group (upper). Five mice per group were examined for western blot analysis (lower). The immunoblot for GAPDH was used as the loading control. Scale bar: 100 µm.

Fig 3: The hepatic accumulation of PPL was highly associated with cholestasis. (A) Representative images for the hepatic accumulation of PPL under different types of hepatitis mediated by the indicated triggers. Protein abundance of PPL measured by western blotting is also shown at the bottom. Equal amounts of protein from 5 mice were combined in each group for western blotting. The immunoblot for GAPDH was used as the loading control. Scale bar: 100 µm. (B) Hepatic PPL expression in vehicle- or ANIT-treated mice examined by immunohistochemical and western blotting analyses. Protein abundances of K19 and ZO-1 examined by western blotting are also shown. Four or 5 mice per group were examined. The immunoblots for GAPDH were used as the loading controls. Scale bar: 100 µm. (C) Hepatic PPL expression levels in bile duct-ligated (BDL, for indicated durations) or sham-operated mice examined by immunohistochemical and western blotting analyses. Immunoblots for K19 and ZO-1 are also shown. Five mice per group were examined. The immunoblots for GAPDH were used as the loading controls. Scale bar: 100 µm. (D) Time course of the induction of hepatic Ppl mRNA expression in wild-type mice after bile duct ligation. Five to 10 mice per group were examined. mRNA expression levels are normalized to Gapdh and are expressed as mean ± SD. The average value in the liver of untreated mice was set to 1.0. ***: P < 0.001 by one-way ANOVA with Dunnett’s test (vs. untreated). (E) Time course of the hepatic PPL accumulation in bile duct-ligated mice examined by western blotting analysis. Three mice per group were examined. The immunoblot for GAPDH was used as the loading control. (F) Representative images for the time course of the hepatic PPL accumulation in bile duct-ligated mice examined by immunohistochemical analysis. Scale bar: 100 µm.

Fig 4: PPL expression in Fxr-/- and CA-fed cholestasis model mice. (A) Hepatic Ppl mRNA expression levels in Fxr+/+ and Fxr-/-mice. Seven or 8 mice per group were examined. Circles and rectangles correspond to individual animals. Horizontal bars represent averages. (B) Hepatic Ppl and Cyp7a1 mRNA expression levels in wild-type mice fed cholic acid (CA) for 7 days at the indicated doses. Five to 9 mice per group were examined. The immunoblot for PPL is also shown. Equal amounts of protein from 5 mice were combined in each group for western blotting. The immunoblot for GAPDH was used as the loading control. (C) Ppl mRNA expression in various tissues following CA feeding for 7 days. Bars for each tissue are grayscaled as indicated (darkened from left to right, tongue to skin). Bars for the liver are flagged with a number sign (#). Four samples per group were examined. (D) Hepatic mRNA expression of plakins, cell junction proteins, and intermediate filaments in wild-type mice fed CA for 7 days. Five to 9 mice per group were examined. mRNA expression levels are represented as values normalized to Gapdh. The average values in Fxr+/+ and control diet-fed mice are set as 1.0. Values represent mean ± SD. Dsg1/2/4, desmoglein-1/-2/-4; Dsc2, desmocollin-2; Dsp, desmoplakin; Plec, plectin; Macf1, microtubule actin crosslinking factor-1; ß/?-Ctn, ß/?-catenin; Gjp1/2, gap junction protein-1/-2; E/N-Cad, E/N-cadherin; Cldn1/2/3, claudin-1/-2/-3; Ocln, occludin; Zo-1, zonula occludens-1; JAM1, junctional adhesion molecule-1; K7/8/18/19, keratin 7/8/18/19. *, **, ***: P < 0.05, 0.01, and 0.001, respectively, by Mann–Whitney U test (A) or one-way ANOVA with Dunnett’s test (B and D [vs. 0%]).

Fig 5: Immunohistochemical localization of PPL in the cholestatic liver. (A) Representative images for the hepatic localization of PPL in mice fed the control or 1% CA-containing diet for 7 days. PPL was detected by red fluorescence. Scale bar: 100 µm. (B) PPL and ZO-1 (left column), or PPL and ?-CTN (right column), were double-stained by red and green fluorescence, respectively. Merged images are shown at the bottom. Arrowheads indicate PPL expression at ZO-1-negative sites (left bottom). Scale bar: 25 µm. (C) PPL and K19 were double-stained by red and green fluorescence, respectively. Signals for PPL were absent in cholangiocytes and hepatocytes of 1% CA-fed Ppl-/- mice. Scale bar: 100 µm. (D) Protein abundance of the selected hepatic structural molecules in the liver of CA-fed mice at indicated doses for 7 days. Four mice per group were examined by western blotting analysis. The immunoblots for GAPDH were used as the loading controls. PV, portal vein; CV, central vein.