FIG. 3.

H-RAS^V12G-dependent regulation of EGFR signaling. (A) Northern blot analyses showing loss of expression of H-RAS^V12G and EGFR pathway components in vivo after doxycycline withdrawal. The time (in hours) after doxycycline withdrawal is shown above the lanes. HB-Egf, heparin-binding EGF-like growth factor; Gapdh, glyceraldehyde-3-phosphate dehydrogenase. (B) RNA in situ hybridization showing Ereg expression during tumor regression. (C) Real-time RT-PCR analysis of H-RAS^V12G, Ereg, Areg, and EGFR expression in melanomas at intervals following doxycycline withdrawal in vivo. The data are from a representative experiment. A second independent experiment showed comparable results. The y axis shows expression relative to time zero expression in the first experiment for each cell line. C590 expresses H-RAS^V12G constitutively. (D) In vitro measurement of Ereg and Areg expression in a series of independently derived Tyr/Tet-RAS melanoma cell lines grown in the presence of doxycycline (Melanoma) and in cultured melanocytes (Mel) (in the presence [left bar] or absence [right bar] of doxycycline). The y axis shows expression relative to that for cultured melanocytes in the absence of doxycycline (rightmost bar).