Rictor shrna

Rictor shRNA was purchased from Addgene (#1853 and #1854, Watertown, MA, USA), and the sequences were as follows: Rictor shRNA1, 5′-CCG GTA CTT GTG AAG AAT CGT ATC TTC TCG AGA AGA TAC GAT TCT TCA CA A GTT TT TTG-3′; Rictor shRNA2, 5′-CGG GCA GCC TTG AAC TGT TTA ACT TCC TGT CAT TAA ACA GTT CAA GGC TGC TTT TTG AAT T-3′. Rictor shRNA plasmids were cotransfected with packing plasmids (psPAX2 and pCMV-VSV-G) into HEK293FT cells. Seventy-two hours after transfection, the supernatant containing the lentivirus particles was collected and added to the cells along with polybrene (10 μg/ml) (#sc-134220 Santa Cruz Biotechnology, Santa Cruz, CA) to generate the Rictor-silenced stable cell line. Twenty-four hours after lentivirus infection, cells were selected in the presence of 1–3 μg/ml puromycin (#A610593, Sangon Biotech, Shanghai, China). The stable clones were then picked, and Rictor expression was validated by western blotting.