Figure 4

Activation of the Akt-mTOR axis in stellate morphogenesis. A) Total cell protein was extracted from A549 cells in rBM 3-D culture exposed to the indicated combinations of TGF-β1 and Col-1. The protein levels of total and phosphorylated Src, Akt, and mTOR were determined using immunoblots. B) Similar to A except that total cell protein was extracted from A549LCvec and A549LCdnSrc cells in rBM 3-D culture exposed to TGF-β1 and Col-1. The protein levels of total and phosphorylated FAK, Akt, mTOR, and p70 S6K were determined using immunoblots. C) A549LC cells were cultured in rBM 3D culture supplemented with TGF-β1 and Col-1 in the presence or absence of Torin-1 (250 nM). Morphogenesis of A549LC cells was monitored for 12 days. Images were captured using phase contrast microscopy. D) The culture condition was identical to that described in part B. Total cell RNA was extracted from A549 cells. The expression of LOX, PAI-1, and Myc was determined using quantitative RT-PCR. Ratios of each gene transcript over the housekeeping gene 36B4 were compared across various culture conditions. A fold change was obtained by setting the values from the control group to one. Mean and standard deviations were obtained from three independent experiments. * and ** indicate a P value < 0.05 and 0.01, respectively.