Gene silencing
PC3 or HEK293T cells were seeded into 6 wells plate at a confluence of 1×106 and then transfected with the siRNA using Lipofectamin RNAi MAX (Thermo Scientific) according to the manufacturer protocol, the following sequences used for siRNA as follows:
FOXO1: FOXO1 (sense: 5’-GGAGGUAUGAGUCAGUAUAUU-3’),
AKT1: AKT1 (sense: 5’-UGCUGUUGACAGUGAGCG-3’),
IGF-1: IGF-1 (sense: 5’-CGCAGGUAACGAUGGGAAAUU-3’),
Control sequence (sense: 5’-UGCCGUUCUUCAACGAGGA-3’).
The siRNA oligonucleotides, together with the corresponding antisense oligonucleotides, were synthesized by Gene Script (Nanjing, China).