Inhibition of ERK signaling augments perifosine-induced cytotoxicity. (A) BCWM.1 cells were cultured with perifosine for 6 hours at doses that range from 2 to 20 μM. Whole cell lysates were subjected to Western blotting using anti–p-ERK, –ERK1/2, –p-c-Raf Ser259, –pan-p-PKC, and α-tubulin antibodies. (B) BCWM.1 cells were cultured for 48 hours with media and with perifosine (5-20 μM) in the absence or presence of 5 and 10 μM of MEK1/2 inhibitor U0126. Cytotoxicity was assessed by the MTT assay. Data represent mean (± SD) of triplicate experiments. (C) BCWM.1 cells were cultured with control media and either perifosine (10 μM), U0126 (5 μM and 10 μM), or the combination for 10 hours. Whole cell lysates were subjected to Western blotting using anti–p-ERK, -ERK1/2, -caspase 9, -caspase 8, -PARP and –α-tubulin antibodies. Horizontal lines have been inserted to indicate a repositioned gel lane. (D) BCWM.1 cells were transduced with 2 Akt shRNA for 48 hours. Mock: control plasmid; 62 and 63 represent shRNA that target 2 different sequences of Akt gene. Whole cell lysates were subjected to Western blotting using anti–p-ERK, -ERK1/2, and α-tubulin antibodies. (E) BCWM.1 cells were cultured with triciribine for 6 hours with doses that ranged from 1 to 10 μM. Whole cell lysates were subjected to Western blotting using anti–p-ERK and α-tubulin antibodies. (F) BCWM.1 was cultured with perifosine (20 μM, 4 hours) in the absence or presence of 25 μM of PI3K inhibitor LY294002. BCWM.1 was cultured with control media, perifosine (20 μM), LY294002 (25 μM, 15 minutes), LY294002 for 15 minutes (1) followed by perifosine for 4 hours (2), or perifosine for 4 hours (1) followed by LY294002 for 15 minutes (2) Whole cell lysates were subjected to Western blotting using anti–p-ERK, -ERK1/2, –p-Akt Ser473, -Akt, –p-c-Raf Ser259, –pan-p-PKC and α-tubulin antibodies.