Plenti cmv puro dest w118 1 vector

4 × 10⁶ 293T cells were seeded in 25-cm² flasks. Transfection was performed to generate lentiviral particles using a four-plasmid system as per the TRC Library Production and Performance protocols, RNAi Consortium, Broad Institute (19 (link)) and as previously described (17 (link)). CDC25C shRNA constructs were purchased from the Sigma MISSION® shRNA Library, and their respective sequences are listed in supplementary table S2. pLKO.1-shRNA scramble (SCR) vector was obtained from Dr. David M. Sabatini through Addgene (plasmid 1864) as previously described (20 (link)). The pLenti CMV Puro DEST (w118-1) vector was obtained from Eric Campeau through Addgene (plasmid 17452). The Ultimate™ ORFs (Invitrogen) for CDC25C and RSK isoforms was obtained from the Johns Hopkins University HiT Center and an LR reaction (Invitrogen) was performed to construct pLenti Puro DEST (w118-1)-CDC25C. All constructs were sequence verified. The ORF clone IDs of the constructs are – IOH14569 (RSK1.a, variant 1), IOH12130 (RSK1.b, variant 2), IOH63248 (RSK2), IOH3648 (RSK3), IOH36120 (RSK4), and IOH14569 (CDC25C).

4 × 10⁶ 293T cells were seeded in 25-cm² flasks, and were transfected to generate lentiviral particles using a four -plasmid system as per the TRC Library Production and Performance protocols, RNAi Consortium, Broad Institute (10 (link)) and as previously described (9 (link)). p90RSK shRNA constructs were purchased from Johns Hopkins University HiT Center, and their respective sequences are listed in supplementary table 2. pLKO.1-shRNA scramble vector was obtained from Dr. David M. Sabatini through Addgene (Addgene plasmid 1864) as previously described (11 (link)). The pLenti CMV Puro DEST (w118-1) vector was obtained from Eric Campeau through Addgene (Addgene plasmid 17452). The Ultimate™ ORFs (Invitrogen) for RSK1-4 were obtained from the Johns Hopkins University HiT Center and an LR reaction (Invitrogen) was performed to construct pLenti Puro DEST (w118-1)-RSK 1-4. All constructs were sequence verified. The ORF clone IDs of the constructs are – IOH46696 (RSK1.a, variant 1), IOH12130 (RSK1.b, variant 2), IOH63248 (RSK2), IOH3648 (RSK3), and IOH36120 (RSK4).

The pLenti CMV Puro DEST (w118-1) vector (Addgene plasmid # 17452) encoding human MDA-7/IL-24 or the empty vector together with pMD2.G (Addgene plasmid # 12259) and psPAX2 plasmid (Addgene plasmid # 12260) were co-transfected into phoenix cells using Fugene HD Promega (Madison, WI). Lentiviral supernatants containing control lentivirus (LV-vec) or lentivirus expressing MDA-7/IL-24 (LV-mda-7) were collected 48 and 72 h after transfection. Day 5 expanded T cells were transduced with LV-vec or LV-mda-7 (2–3 × 10⁶ cells/mL in 6-well plate at MOI of 5 in the presence of 6 μg/mL polybrene) under centrifugation at 2,000 g for 90 mins at 32 °C. Transduced T cells were cultured in the presence of IL-7 and IL-15 overnight before collection for studies.