mTOR kinase activity remains high when IPF fibroblasts are cultured on collagen matrix.

<p>A) Left, 2×10⁵ serum starved control lung (n = 11) and IPF fibroblasts (n = 10) were cultured on polymerized collagen in serum free medium for 24 h and ser 2448 p-mTOR (p-mTOR) levels were measured by Western analysis. Shown is a Western analysis for p-mTOR levels in all IPF and control fibroblasts grown in serum free medium on polymerized collagen. GAPDH was used as a loading control. Right, Shown is a box-and-whisker plot of p-mTOR protein expression in 11 control and 10 IPF fibroblasts normalized to GAPDH on collagen matrix. <i>p</i><0.001 IPF versus control fibroblasts. B) Upper, randomly selected 2×10⁵ of 3 control or of 3 IPF fibroblasts were cultured on polymerized collagen in serum free medium as a function of time and ser 2448 p-mTOR levels were measured. GAPDH was used as a loading control. Lower, shown is the p-mTOR/GAPDH protein expression ratio in randomly selected control and IPF fibroblasts as a function of time. <i>p</i><0.01 IPF versus control fibroblasts at 24 h. C) Upper, control and IPF fibroblasts (n = 5 each) were cultured in the presence of various doses of rapamycin on collagen gel for 24 h and p-mTOR level was then measured. Shown is the representative p-mTOR protein level in control and IPF fibroblasts on polymerized collagen. GAPDH level was used as a loading control. Lower, p-mTOR/GAPDH expression ratio in control (n = 5) and IPF fibroblasts (n = 5) in the presence of various doses of rapamycin on collagen are shown. <i>p</i> = 0.01 versus DMSO control. <i>p</i><0.01 versus DMSO control as indicated. Cells were randomly selected for this assay. D) IPF fibroblasts infected with adenovirus expressing dominant negative Akt (DA) or empty vector GFP were cultured on collagen gel for 24 h in serum free medium and p-mTOR, Akt, phosphorylated Akt and GAPDH levels were measured. IPF fibroblasts expressing enhanced p-mTOR level was selected to examine whether Akt inhibition suppresses p-mTOR level (lane 5 in upper panel of IPF in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094616#pone-0094616-g002" target="_blank">Fig 2A</a>). This assay was repeated twice. E) Control fibroblasts infected with adenovirus expressing hyperactive Akt (HA) or empty vector (GFP) were cultured on collagen for 24 h in serum free medium and p-mTOR, total Akt, phosphorylated Akt and GAPDH levels were measured. Control fibroblasts expressing low p-mTOR level were selected to examine whether hyperactive Ak increases mTOR kinase activity (lane 3 in upper panel of control fibroblasts in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094616#pone-0094616-g002" target="_blank">Fig 2A</a>). This assay was repeated twice.