FIG. 7.

Complementation of the CSF-1R[809F] mitogenic and signaling defect by v-Src:ER. (A) Activation of the MAP kinases and induction of c-Myc and cyclin D1 by v-Src:ER. NIH 3T3 cells expressing v-Src:ER were cultured in DSFM for 2 days and treated with 500 nM 4-HT for various lengths of time. Expression of c-Myc, cyclin D1, activated p42 and p44 MAP kinases, and v-Src:ER was assessed by Western blotting. (B) Proliferation of v-Src:ER-expressing cells. NIH 3T3 cells expressing the CSF-1R[809F] and v-Src:ER were cultured in DSFM for 24 h and then either left untreated (NT) or treated with 10% FCS, 300 nM CSF-1 (gray bars), 4-HT (0.05 to 1 nM, open bars), or CSF-1 plus 4-HT (solid bars). Cell proliferation was determined by measuring the incorporation of [methyl-³H]thymidine over a period of 48 h. (C) Effect of inhibition of MEK activity on the induction of c-Myc by v-Src:ER. NIH 3T3 cells expressing CSF-1R[809F] and v-Src:ER were cultured in DSFM for 36 h, treated with 100 μM PD098059 for 1 h, and then stimulated with 50 nM 4-HT for 4 h. Expression of c-Myc was assessed by Western blotting. (D) Effect of inhibition of MEK activity on the induction of cyclin D1 by v-Src:ER. NIH 3T3 cells expressing v-Src:ER were cultured for 36 h in DSFM, treated with DMSO (solvent control) or 100 μM PD098059 for 40 min, and then stimulated with various concentrations of 4-HT (0 to 2 nM) for 24 h. Expression of cyclin D1 was assessed by Western blotting.