MTB/TRAS short-term induction, tumor, and TGFbeta treated NMuMG cells

To test whether SVD regression is sufficiently sensitive to detect activation of a secondary, endogenous pathway as it occurs following ectopic manipulation of a strong primary pathway by focusing on the relationship between the Ras and TGFβ signaling pathways. Keywords: Time course, Affymetrix, Bitransgenic MTB/TRAS mice in an FVB/N background were generated by crossing MTB and TRAS mice. To induce oncogenic v-H-Ras expression, 6-week old MTB/TRAS female mice were administered 2 mg/ml doxycycline with 5% sucrose in their drinking water. Mammary tissue was harvested at different post-induction time points and snap frozen. To generate Ras-driven tumors, MTB/TRAS mice were administered 0.012 mg/ml doxycycline in their drinking water and monitored for tumor formation. Mice were sacrificed when tumors reached ~1 cm and tissue was snap frozen. The non-transformed murine mammary epithelial cell line, NMuMG, was cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% bovine calf serum, 1% penicillin/streptomycin, and 2 mM L-glutamine. For TGFβ treatment, cells were cultured in low serum medium (0.5%) overnight followed by treatment with 5 ng/ml TGF-β1 or TGF-β3 (Sigma). After 24 hr, RNA and protein were harvested for microarray hybridization or biochemical analysis.