Pcmv6 mammalian expression vector

All eIF4E, eIF4G (including the Y624A, L629A and L630A binding mutant) and 4EBP1 mutant cDNAs were synthetized and obtained from IDT (Integrated DNA Technologies). eIF4E and eIF4G^604–646 were cloned into NanoBit plasmids using the NanoBit PPI starter system (Promega) using XhoI/EcoRI and NheI/EcoRI cloning sites respectively. 4EBP1 mutants were cloned into pCDNA3.1 vector DNA (Thermo Fisher Scientific) harbouring a C-terminal 3× FLAG tag via NheI/BamHI sites to allow mammalian cell overexpression. For bacterial expression, 4EBP1 mutants were cloned into pGEX6P1 using BamHI/EagI cloning sites. GFP and v-Myc coding sequence residing in a pCMV6 mammalian expression vector were obtained from Origene. The bicistronic luciferase reporter construct pcDNA3-rLuc-polIRES-fLuc was purchased from Addgene. PP242, Torin1, Rapamycin, 4EGi-1 and 4E1RCat were purchased from Tocris Bioscience, whilst all other chemicals unless otherwise stated were purchased from Selleck Chemicals. siRNAs targeting either 4EBP1 (ON-TargetPlus Human EI4EBP1, J-003005-12-0005) or non-targeting control (ON-TargetPlus Non-targeting pool, D-001810-10-05) were purchased from DHARMACON.

For ABCB1 overexpression studies, cells were dual transfected with pCMV6-AC-GFP (empty vector) and pCMV6-AC-ABCB1 (GFP-tagged ABCB1) using Lipofectamine 3000 (Life Technologies). The wild type ABCB1 expression vector consists of full-length ABCB1 (MDR1) cDNA within the pCMV6 mammalian expression vector (Origene Technologies). For FOXO3a overexpression studies, cells were dual transfected with pEGFP-N1 (empty vector) and pEGFP-N1-FOXO3a using Lipofectamine 3000. The fusion GFP-tagged FOXO3a-WT encoding vector was constructed by subcloning the full-length FOXO3a cDNA (Clontech, Palo Alto, CA). Gene-specific methods used for transient transfections were described previously [78 (link), 79 (link)]. For ABCB1 silencing, cells were transfected with scramble siRNA (FITC-conjugate) or human-specific ABCB1 (MDR1) siRNA duplexes (Santa Cruz Biotechnology) using Lipofectamine 3000 [80 (link)]. For FOXO3a silencing, cells were transfected with stealth scramble or FOXO3a siRNAs (Santa Cruz Biotechnology) using Lipofectamine 3000 [81 (link)]. For TUBB3 silencing, cells were transfected with scramble or TUBB3 siRNA oligos (Ambion, Austin, TX) using siLentFect (Bio-Rad) [82 (link)]. Transfectants were subjected to drug treatment and/or assays after 48 hr of transfection. RNAi and transient gene transfection efficiencies and cell morphology are presented in Supplementary Figure 7.