Rapamycin potentiates the growth inhibitory effects of TGFβ.

(A) The indicated cell lines were treated with 2.5 ng/ml TGFβ, 100 nM rapamycin, or TGFβ + rapamycin (T + R) for one week until small colonies of cells formed. Representative images of treatment effects on cell-cell adhesion within colonies are presented. (B) The indicated cell lines were treated with or without 2.5 ng/ml TGFβ for 96 hours and cell extracts were analyzed by immunoblot. (C) PyMT, TβRIIflx/flx cells were treated for one hour with or without 2.5 ng/ml TGFβ and phosphorylation of Smads 2 and 3 and the ribosomal S6 protein were analyzed by immunoblot. (D–F) Proliferation, as measured by [3H]-thymidine incorporation, of the indicated cell lines after treatment for 24 hours [(D), (E), (F) left panel], or for 48 hours [(F) right panel]. (G, H) Immunoblot analysis of lysates from the indicated cell lines after 24 hours of the specified treatment for 24 hours (G) or either 1 or 24 hours (H). (I) Proliferation, as measured by [3H]-thymidine incorporation, of the indicated cell lines after 24 hours of the specified treatment. (J) PyMT, TβRIIflx/flx cells were treated with the indicated combinations of a TβRI kinase inhibitor and rapamycin at different concentrations for 24 hours. Asterisks indicate that the specified TβRI kinase inhibitor concentration reversed rapamycin-mediated cell cycle arrest in a statistically significant manner. Values represent the mean, and error bars are the standard deviation of triplicate measurements. * Denotes P<0.05 as determined using the unpaired Student's t test.