Volume 114, Issue 6, Pages (June 1998)

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Volume 114, Issue 6, Pages 1287-1295 (June 1998) Overexpression of pp60c-src elicits invasive behavior in rat colon epithelial cells Susan E. Pories, Donald T. Hess, Karen Swenson, Margaret Lotz, Runna Moussa, Glenn Steele, David Shibata, Kimberly M. Rieger- Christ, Ian C. Summerhayes Gastroenterology Volume 114, Issue 6, Pages 1287-1295 (June 1998) DOI: 10.1016/S0016-5085(98)70435-4 Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 1 In vitro protein kinase activity of pp60c-src performed in the presence of the exogenous substrate casein. Lane 1, FRC/TEX parental cell line; lane 2, FRC/TEXc-src/C; lane 3, FRC/TEXc-src/D; lane 4, FRC/TEXc-src/9; lane 5, FRC/TEXv-src/C. Lane 5 represents a shorter exposure than lanes 1–4 to provide good resolution. The relative kinase activities are represented in histogram format below and assessed after a standard exposure time. Gastroenterology 1998 114, 1287-1295DOI: (10.1016/S0016-5085(98)70435-4) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 2 Western blot analysis of avian pp60c-src expression after immunoprecipitation with a panreactive src antibody (Mab 327) probed with an avian-specific src antibody (ECI0). Lane 1, FRC/TEX parental colon epithelial cell line; lane 2, FRC/TEXc-src/9; lane 3, FRC/TEXc-src/B; lane 4, FRC/TEX/hygro/A control; lane 5, FRC/TEXc-src/ C; lane 6, FRC/TEXc-src/D; lane 7, FRC/TEXv-src/C. Black arrowhead shows migrational level of avian c-src; open arrowhead denotes immumoglobulin heavy chain. Gastroenterology 1998 114, 1287-1295DOI: (10.1016/S0016-5085(98)70435-4) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 3 Phosphotyrosyl protein profiles in total cell lysates of colon lines. Lane 1, FRC/TEX parental cell line; lane 2, FRC/TEXc-src/9; lane 3, FRC/TEXc-src/C; lane 4, FRC/TEXc-src/D; lane 5, FRC/TEXv-src/C, where one-fifth of the total protein amount was loaded in this lane. Gastroenterology 1998 114, 1287-1295DOI: (10.1016/S0016-5085(98)70435-4) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 4 Morphology of colon epithelial cell lines after introduction and expression of different src constructs. (A) FRC/TEX parental cell line; (B) FRC/TEXc-src/C; (C) FRC/src527/P; (D) FRC/TEXv-src. Gastroenterology 1998 114, 1287-1295DOI: (10.1016/S0016-5085(98)70435-4) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 5 Colony growth in soft agar. (A) FRC/TEX/hygro/A control cell line; (B) FRC/TEXc-src/C; (C) FRC/TEXv-src. Gastroenterology 1998 114, 1287-1295DOI: (10.1016/S0016-5085(98)70435-4) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 6 In vitro (A) migration and (B) invasion assays performed using a panel of rat colon epithelial cells expressing different levels of the c-src proto-oncogene. The parental cell line transfected with the hygromycin gene (FRC/TEX) or the viral src oncogene (FRC/TEXv-src) were used as control cell lines in each assay. FRC/TEX clones 9, C, and D represent three colon epithelial cell transfectants overexpressing c-src. Gastroenterology 1998 114, 1287-1295DOI: (10.1016/S0016-5085(98)70435-4) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 6 In vitro (A) migration and (B) invasion assays performed using a panel of rat colon epithelial cells expressing different levels of the c-src proto-oncogene. The parental cell line transfected with the hygromycin gene (FRC/TEX) or the viral src oncogene (FRC/TEXv-src) were used as control cell lines in each assay. FRC/TEX clones 9, C, and D represent three colon epithelial cell transfectants overexpressing c-src. Gastroenterology 1998 114, 1287-1295DOI: (10.1016/S0016-5085(98)70435-4) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 7 Western blot analysis of total cell lysates probed with antibodies to E-cadherin and α-, β-, and γ-catenin. Lane 1, FRC/TEX control parental rat colon epithelial cell line; lane 2, FRC/TEXc-src/9; lane 3, FRC/TEXc-src/C; lane 4, FRC/TEXc-src/D; lane 5, FRC/TEXv-src. Gastroenterology 1998 114, 1287-1295DOI: (10.1016/S0016-5085(98)70435-4) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 8 Immunoprecipitation of β-catenin from the panel of colon epithelial transfectants (A) probed in Western blot analysis with antiphosphotyrosine antibody and (B) stripped and reprobed with anti–β-catenin antibody. Lane 1, FRC/TEX parental cell line; lane 2, FRC/TEXcsrc/9; lane 3, FRC/TEXc-src/C; lane 4, FRC/TEXc-src/D; lane 5, FRC/TEXv-src; lane 6, FRC/TEX total cell lysate, positive control for phosphotyrosine blot. Gastroenterology 1998 114, 1287-1295DOI: (10.1016/S0016-5085(98)70435-4) Copyright © 1998 American Gastroenterological Association Terms and Conditions