PLD inhibitors block mTOR signaling pathway and exogenous PA abrogates the effect of PLD inhibitors on mTOR signaling and proliferation of PKD cells.

<p>(A) PLD inhibitors affect mTOR signaling in a dose-dependent way. Western blots analyzing the expression of phospho-Akt, Akt, phospho-S6K, S6K, phospho-S6, S6, phospho-4EBP1, 4EBP1 and GAPDH either upon treatment with the indicated concentration of PLD1-/2- inhibitor for 48 h or without treatment. (B) PKD cells were plated for 24 h and then shifted to medium containing 0.5% serum. 1% 1-BtOH or 1% <i>tert</i>-BtOH was added for 2 h, 20 µM honokiol was added for 20 h. Western blots analyzing the expression of phospho-S6K, S6K, phospho-Akt and Akt. (C, D) Exogenous PA stimulated mTOR signaling in a time-dependent way, but not PLD activity. Human PKD cells were cultured under either normal culture medium (5% FBS) or starvation culture medium (0.5% FBS). After 24 h starvation, cells were treated with 100 µM PA for 20, 40 and 60 minutes. Whole cell lysates were analyzed by (C) western blots to detect the expression of phospho-mTOR, mTOR, phospho-S6K, S6K, phospho-Akt, Akt and GAPDH. (C) Whole cell lysates were determined by Amplex Red PLD kit to measure the PLD activity. (E) Exogenous PA impairs the effect of PLD inhibitors on cell growth. Cells were pretreated with either 10 µM PLD1 inhibitor or 20 µM PLD2 inhibitor for 48 h, and then with 100 µM PA for the indicated time. DNA synthesis was measured by using the BrdU assay. An experiment which is representative of three independent experiments is shown. Data are expressed as mean ± SD and were analyzed by one-way ANOVA. * p< 0.05, ** p< 0.01.