Volume 41, Issue 5, Pages 808-814 (November 2004) Enhanced epidermal growth factor receptor activation in human cholangiocarcinoma cells  Jung-Hwan Yoon, Geum-Youn Gwak, Hyo-Suk Lee, Steven F. Bronk, Nathan W. Werneburg, Gregory J. Gores  Journal of Hepatology  Volume 41, Issue 5, Pages 808-814 (November 2004) DOI: 10.1016/j.jhep.2004.07.016 Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 1 The enhanced and prolonged EGFR activation in cholangiocarcinoma cells. KMBC, Witt and HepG2 cells were cultured in the presence and absence of EGF (50ng/ml) for the indicated time intervals. Equivalent amounts of protein were immunoblotted (IB) with anti-phosphotyrosine antibody (PhoTyr), anti-EGFR antibody, antisera specific for the phosphorylated forms of p42/p44 MAPK (Pho-p42/44) and total p42/p44 MAPK (total-p42/p44). Journal of Hepatology 2004 41, 808-814DOI: (10.1016/j.jhep.2004.07.016) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 2 EGFR ubiquitination occurs in cholangiocarcinoma cells. KMBC, Witt and HepG2 cells were stimulated with EGF (50ng/ml) for the indicated time intervals. Immunoprecipitation of the EGFR from cell lysates was performed followed by immunoblot analyses using either anti-ubiquitin or anti-EGFR antibody. Journal of Hepatology 2004 41, 808-814DOI: (10.1016/j.jhep.2004.07.016) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 3 EGFR internalization is defective in cholangiocarcinoma cells. (A) Cells were stimulated with EGF (50ng/ml) for the each indicated time intervals. After fixation, cells were incubated with anti-EGFR antibody followed by FITC-conjugated secondary antibody staining. Analysis was performed by confocal microscopy at ×400 magnification. (B) Cell-surface molecules were biotinylated, followed by incubation with EGF (50ng/ml) for the indicated time intervals. The EGFR was immunoprecipitated from cell lysates, and immunoblotted with horseradish peroxidase-conjugated streptavidin. Journal of Hepatology 2004 41, 808-814DOI: (10.1016/j.jhep.2004.07.016) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 4 EGFR kinase inhibitors attenuate cholangiocarcinoma cell growth. KMBC and HepG2 cells cultured in 96-well plates were treated with EGF (50ng/ml) or the EGFR kinase inhibitor, AG1478 (2μM) or ZD1839 (1.5μM). At each indicated time point, an MTS assay (Promega) was performed according to the manufacturer's instruction. Data were expressed as mean±SD of percent change of optical densities as compared to that of the day 0. *P<0.05 vs. control. Journal of Hepatology 2004 41, 808-814DOI: (10.1016/j.jhep.2004.07.016) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 5 EGFR kinase inhibitors reduce COX-2 expression levels in cholangiocarcinoma cells. KMBC and HepG2 cells were incubated in the presence of EGF (50ng/ml) with or without EGFR kinase inhibitor, AG1478 (2μM) or ZD1839 (1.5μM). Cells were lysed after 24h, and immunoblot analysis was performed using anti-COX-2 antisera. Immunoblot analysis using anti-actin antisera was performed as a control for protein loading. Journal of Hepatology 2004 41, 808-814DOI: (10.1016/j.jhep.2004.07.016) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 6 COX-2 inhibitor attenuates cholangiocarcinoma cell growth. KMBC cells were cultured in 96-well plates either in the presence or absence of the COX-2 inhibitor, NS398 (NS, 100μM). At each indicated time point, an MTS assay was performed. Data were expressed as mean±SD of percent change of optical densities as compared to that of the day 0. *P<0.05 vs. control. Journal of Hepatology 2004 41, 808-814DOI: (10.1016/j.jhep.2004.07.016) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 7 PgE2 prevents EGFR kinase inhibitor-induced growth suppression in cholangiocarcinoma cells. KMBC cells cultured in 96-well plates were treated with the EGFR kinase inhibitor, AG1478 (2μM) or ZD1839 (1.5μM) either in the presence or absence of PgE2.At each indicated time point, an MTS assay was performed. Data were expressed as mean±SD of percent change of optical densities as compared to that of the day 0. *P<0.05 vs. PgE2 0μg/ml. Journal of Hepatology 2004 41, 808-814DOI: (10.1016/j.jhep.2004.07.016) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions