Lentiviral-mediated Knockdown of EIF3D and RHEB

The knockdown study was performed using lentiviral-mediated RNA interference. The lentiviral vectors with the sequences for the shRNAs were purchased from the Erasmus Center for Biomics. The shRNA sequences targeting EIF3D and RHEB were 5′-CCGGCCTCAGACATACTCCATAGATCTCGAGATCTATGGAGTATGTCTGAGGTTTTTG-3′, respectively. A scrambled shRNA (shscramble) was used as control, as previously described [119 (link)]. For the lentivirus production, a four plasmid system was used for the transient transfection of 293T cells as previously described [120 (link), 121 (link)], followed by concentration with Amicon Ultra Centrifugal Filters-100K Units (Merck Millipore). The titers of the concentrated lentiviruses were determined after infection of HT1080 cells with serial dilutions of the viral stock. The titers were estimated at 5×10⁸ – 10⁹ infection units (IU)/ml. The lentiviruses were then used for the transduction of the target cell line T24M. As control T24M cells transduced with a lentivirus for shscramble was used. The knockdown effect was evaluated at the RNA level by real-time PCR and at the protein level by Western blot analyses as described in the following sections.