4e1rcat

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.

The cardiac muscle cell line HL-1 (a generous gift from Dr. William Claycomb, - Louisiana State University Medical Center) is an atrial cardiomyocyte tumor lineage originally derived from female AT-1 mouse [25] (link). Cells were cultured in a 37°C incubator in the presence of 5% CO₂ in complete Claycomb medium using flasks pre-coated with 12.5 µg/ml bovine fibronectin (Sigma) in 0.02% gelatin solution (Sigma, St Louis, MO) as described previously [25] (link), [28] (link). Confluent cells were washed with 1X phosphate buffered saline (PBS), and incubated in serum-free Claycomb medium prior to treatments with bovine insulin (100 nM:12 hr), rapamycin (10 nM:12 hr), p70S6K1 inhibitor PF-4708671(2 µM;12 hr), or 4E1RCat, an inhibitor of cap-dependent translation (5 µM;12 hr). Insulin was purchased from Sigma-Aldrich Inc., rapamycin from Cell signaling Technology, and PF-4708671 and 4E1RCat were from Tocris Bioscience. At the end of treatments with different agents, medium was removed, cells were rapidly cooled with ice-cold PBS, collected using cell scrapers followed by centrifugation at 3500 rpms for 5 minutes at 4°C, and cell pellets were flash frozen using liquid nitrogen. Cell pellets were stored in a -80°C ultra-freezer until further processing. All treatments were performed at least in triplicate.