FIG. 4.

Evaluation of cholangiocyte proliferation and interactions with other cell types. (A) Liver tissue sections from WT, MDR2^−/−, and MDR2^−/− plus LSCEVs were stained with CK-19, a marker of cholangiocytes, to evaluate biliary mass. Biliary mass was measured by quantifying the amount of positive staining by the total area. This quantification is displayed as mean percentage of biliary mass ± SEM. *P < 0.05 versus WT; ^#P < 0.05 versus MDR2^−/−. (B,C) Macrophage infiltration (marker F4/80) and liver fibrosis (marker α-SMA) around ductular reaction areas (marker CK-19) were detected in MDR2^−/− mouse livers when compared with WT control by double-staining immunohistochemistry analysis. Multiple antigen labeling in the same tissue section was done using the VECTASTAIN systems (Vector Laboratories, Inc., Burlingame, CA). Specific enzyme substrates were incubated in sections to develop contrasting optimal color (F4/80/α-SMA, brown; CK-19, red). The representative images from four separate experiments are displayed. (D) Cholangiocyte proliferation was measured with real-time quantitative PCR for the proliferation markers PCNA and Ki-67. Data are normalized to WT and presented as mean ± SEM. *P < 0.05 versus WT; ^#P < 0.05 versus MDR2^−/−. (E) Total liver senescence was measured by real-time quantitative PCR for the senescence markers p16 and p21. Data are shown as mean ± SEM. *P < 0.05 versus WT; ^#P < 0.05 versus MDR2^−/−.