Effects of mTOR inhibitors on cholangiocyte apoptosis in

<p>Apoptosis was determined following the treatment of PCK cholangiocytes with rapamycin (100 ng/ml), everolimus (100 nM) and NVP-BEZ235 (500 nM) as described in the Materials and Methods. Flow cytometric analysis showed that 24-hour treatment with rapamycin and everolimus significantly induced apoptosis in PCK cholangiocytes, while it was significantly inhibited by the treatment with NVP-BEZ235 (A). NVP-BEZ235 significantly reduced the expression of cleaved caspase 3 in PCK cholangiocytes that was determined using Western blot analysis (B). The results of the semiquantitative analysis of Western blotting are shown in C. Flow cytometric analysis showed that silencing of caspase 3 using siRNA in PCK cholangiocytes resulted in the reduction of the percentage of apoptotic cells under normal and H₂O₂-treated conditions (D). The analysis with quantitative real-time PCR showed that treatment with NVP-BEZ235 significantly increased the expression of bcl-2 mRNA in PCK cholangiocytes (E). *, <i>p</i><0.01; **, <i>p</i><0.05 (vs. untreated control).