Dual silencing of SAG and PHLPP1/DEPTOR

The lenti-virus-based siRNA knockdown of SAG (Lt-SAG, 5’-GAGGACUGUGUUGU GGUCU-3’), along with scrambled siRNA control (Lt-Con, 5’-AUUGUAUGCGAUC GCAGAC-3’) was performed as described [7 (link)]. For double silencing, cells were infected with Lt-Sag or Lt-Con for 48 to 72 hrs in 60-mm dishes. Cells were then split into 60-mm dishes and transiently transfected with si-Con or Si-PHLPP1 (siRNA pools from Santa Cruz Biotechnology, Santa Cruz, CA) or Si-DEPTOR (5’-GCCATGACAATCGGAAATCTA-3’) using Lipofectamine 2000 (Life Technology, Carlsbad, CA). Forty-eight hours later, cells were harvested for proliferation, clonogenic, migration and soft agar assay.