(A and B) MLN4924-IR induced G2 arrest and accumulation of WEE1/p21/p27. Subconfluent cells were treated with MLN4924 (DU145 at 200 nM and PC3 at 150 nM) or IR (4 Gy) or MLN4924+IR, followed by cell cycle profile analysis (A), and IB analysis (B), using antibodies against cullin1, WEE1, p21, p27, p-H3, t-H3, and cyclin B1, with GAPDH as a loading control. (C) MLN4924-IR had little effect on the transactivation of WEE1/p21/p27. DU145 and PC3 cells were treated with IR (4 Gy) or MLN4924 (200 nM for DU145 and 150 nM for PC3) + IR (4Gy) for 6 hours, then subjected to real-time PCR for WEE1/p21/p27 with GAPDH as an internal control (n = 3). Shown is mean ± SEM, (n = 3): *P < 0.05, **P < 0.01, ***P < 0.0001. MLN, MLN4924; IR, irradiation.