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. 2014 Apr 24;9(4):e96023. doi: 10.1371/journal.pone.0096023

Figure 7. RAP inhibits mTOR signaling in HCECs in a β-catenin/TCF transcription-independent manner in vitro.

Figure 7

HCEC lines stably transformed with shAPC (HCEC/ptripzshAPC) or shScramble (HCEC/ptripzshSCR) were treated with Doxycycline (DXC) at 1 µg/ml to induce shRNA expression for 48 hr or untreated (-). The cells were then treated with RAP (0.001–1 µM) or vehicle (−) for 24 hr in the presence of Doxycycline under serum starving condition (0.2% serum). Total cell lysates were analyzed by Western blotting for phospho-S6 and phospho-mTOR. β-actin protein levels served as loading control (A). Significant decrease in phospho-S6 and phospho-mTOR levels was observed in cells treated with RAP. Expression of selected β-catenin/TCF-regulated target genes AXIN2, NKD1 and IRS1 were analyzed in the same cells by real time PCR (B–D). The results represent the mean ± SEM; n = 5 for each treatment group. There were no statistical differences between groups.