Supplemental Figure S5.

Activation of Yes-associated protein (YAP), Notch1, and mammalian target of rapamycin (mTOR) in human intrahepatic cholangiocarcinoma (iCCA) samples. A: Representative immunohistochemical pattern of connective tissue growth factor (CTGF), a YAP target, in sections from the nontumorous surrounding liver (ST) and two human iCCAs (iCCA1 and iCCA2; the same tumor samples are stained with other antibodies in Figure 8). In ST, no staining was detected for CTGF in normal hepatocytes and biliary cells (arrow). Both tumors exhibited strong immunoreactivity for cytoplasmic CTGF. B: Levels of Notch1, YAP, phosphorylated/inactivated YAP (S127), and phosphorylated/activated mTOR (S2448) in normal cholangiocyte (NHC-SS and C324) and iCCA (HuccT1, RBE, and KKUM-213) cell lines as determined by Western blot analysis. β-Actin was used as a loading control. C: Quantitative real-time RT-PCR analysis of Notch1 (HES1, HEY1), YAP (CYR61), and mTOR (SLC38A1, SLC1A5, and RAPTOR) mRNA levels in normal cholangiocyte (NHC-SS and C324) and iCCA (HuccT1, RBE, and KKUM-213) cell lines. Gray bars indicate normal cholangiocyte cell lines, and black bars indicate iCCA cell lines. Quantitative values were calculated using the PE Biosystems analysis software and expressed as number target (N target). N target = 2−ΔCt, wherein the ΔCt value of each sample was calculated by subtracting the mean Ct value of the gene of interest from the mean Ct value of the GAPDH gene. Scale bars: 50 μm (A, left panel); 100 μm (A, middle and right panels). Original magnification: ×400 (A, left panel); ×200 (A, middle and right panels).