mTOR and PI3 Kinase Inhibitor Synergy Figure 5. BEZ235 alone or in combination with temsirolimus induces G1 cell cycle arrest and p27 expression. A–B, AN3CA (A) or Hec50co (B) cells were treated for 24 hrs or 72 hrs, respectively, with vehicle (DMSO) or BEZ235 (1 nM–100 nM) in the presence or absence of 1 nM temsirolimus. Cell cycle distribution was analyzed by flow cytometry and the percentage of cells in G1 determined. C, AN3CA cells were treated for 24 hrs with vehicle (DMSO) or ZSTK474 (1 mM) in the presence or absence of 1 nM temsirolimus. Cells were analyzed as in A. D, Expression of the cyclin-dependent kinase inhibitor p27 was assessed by Western blotting 24 hrs after the indicated treatments. doi:10.1371/journal.pone.0026343.g005 PLoS ONE | www.plosone.org 8 October 2011 | Volume 6 | Issue 10 | e26343