mTOR and PI3 Kinase Inhibitor Synergy Figure 6. Combination treatment induces autophagy and PARP cleavage. (A) Expression of the autophagy marker LC3 was assessed by Western blotting after the indicated treatments for 24 hrs. Arrows denote full-length LC3-I (16 kDa) and cleaved LC3-II (14 kDa). (B) PARP cleavage was assessed by Western blotting after the indicated treatments for 48 hrs–72 hrs. Arrows denote full-length PARP (116 kDa) and cleaved PARP (89 kDa). doi:10.1371/journal.pone.0026343.g006 resistant to temsirolimus alone. These findings are consistent with a recent study in melanoma cells in which dual treatment with the PI3K inhibitor PI-103 and rapamycin reversed compensatory Akt phosphorylation and induced cell cycle arrest, and xenograft studies demonstrated reduced tumor growth with this combination strategy [33]. We extend these findings herein to define a potential mechanism by which the combination therapy promotes cell death. PLoS ONE | www.plosone.org 9 October 2011 | Volume 6 | Issue 10 | e26343