Sc 373910

Human PC3 and DU145 cell lines were purchased from ATCC (Manassas, VA), cultured in DMEM high glucose medium (Hyclone, Logan, UT) with 10% Fetal bovine serum (FBS, Atlanta Biologicals, Atlanta, GA), 100 U/ml penicillin, and 100 mg/ml streptomycin in a humidified incubator at 37°C and 5% CO2. Cells were routinely passaged and when they were 80–90% confluent, transfection was carried out using SMARTvector 2.0 Lentivirus ShControl (non-targeting) and ShAkt1 (ACGCTTAACCTTTCCGCTG) from Dharmacon (Lafayette, CO), followed by selection with puromycin (0.6 ng/ml, Sigma Millipore, St. Louis, MO). Primary antibodies against Akt1 (Cat #2938), p^Ser473Akt (Cat #4060), p^Thr308Akt (Cat #2965), pFoxO1/3a (Cat #9464), pFoxO3a (Cat #9465), FoxO3a (Cat #2497), FoxO1 (Cat #2880), pSmad2/3 (Cat #8828), Smad2/3 (Cat #8685), Snail (Cat #3879), E-cadherin (Cat #3195), and N-cadherin (Cat #4061) were purchased from Cell Signaling (Danvers, MA). Nodal antibodies (Cat # SC-28913 and SC-373910) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). β-actin antibody (Cat #A5441) was purchased from Sigma (St. Louis, MO). Triciribine (TCBN) and SB505124 were purchased from Selleckchem (Houston, TX), and FoxO1/3a inhibitor (AS1842856) was purchased from Calbiochem (San Diego, CA). All other reagents were purchased from Fisher Scientific (Hanover Park, IL).