Lentiviral plasmid vector construction and viral production

For constructing lentiviral vectors expressing WT Rheb or constitutively active Rheb-Q64L mutant, two primers (forward: 5’-caccatggactacaaagaccatgacggt-3’; reverse: 5’-tcacatcaccgagcacgaagactttccttg-3’) were used to amplify the respective DNA fragments. The fragments were first sub-cloned into pENTR/D-TOPO vector (Invitrogen, Carlsbad, CA) and subsequently to the pLV7 destination vector that harbor a puromycin resistance gene via Gateway cloning to generate the pLV7-CMV-Rheb expression vector. Viral particles were generated following standard protocols in 293T cells, as described previously[76,77]. Viral particles were concentrated using Lenti-X concentrator (Clontech Lab, Mountain View, CA). Cells were infected and selected with puromycin to generate stable expression cell lines. For lentiviral shRNA vectors, we followed the procedures described in our previous study[30]. Non-specific shRNA construct (GCAACAAGATGAAGAGCAC) was described in that study. Five mTor targets were designed and tested for knockdown efficiency. The target sequences for knockdown in MMH-D3 cells were GTGGAGCCCTACAGGAAGT and GTGCTACATTGGCTGGTGT, and those for 3T3-L1 cells were GCCACACCGTGATGGAAGT and GTGCTACATTGGCTGGTGT. Viral particles were prepared as above and used to infect MMH-D3 cells. Two days post-infection, cells were selected with 2 μg/ml puromycin and stable cells lines were used for rhythm assays.