pNGL, pΙκΒαDN, and pCAG.LUC (#74409) have been described elsewhere8 (link),25 (link),33 (link). Lentiviral shRNA pools (Santa Cruz) are described in Supplementary Table 8 . A pMIGR1-based (#27490) bicistronic retroviral expression vector was generated by replacing eGFP sequences with puromycin resistance gene (#58250). KrasG12C,Chuk, Ikbkb, Ikbke, and Tbk1 cDNAs were cloned via reverse transcriptase-PCR (RT-PCR) from LLC or MC38 RNA using specific primers (Supplementary Table 6 ) and were subcloned into peGFP-C1 (Takara, Mountain View, CA). eGFP, eGFP.KrasG12C, eGFP.Chuk, eGFP.Ikbkb, eGFP.Ikbke, and eGFP.Tbk1 cDNAs were subcloned into the new retroviral expression vector (#58249, #64372,# 87033, #58251, #87444, and #87443, respectively). Retroviral particles were obtained by co-transfecting HEK293T cells with retroviral vectors, pMD2.G (#12259), and pCMV-Gag-Pol (Cell Biolabs, San Diego, CA) at 1.5:1:1 stoichiometry using CaCl2/BES. After 2 days, culture media were collected and applied to cancer cells. After 48 h, media were replaced by selection medium containing 2–10 μg/mL puromycin. Stable clones were selected and subcultured11 (link). For stable plasmid/shRNA transfection, 105 tumor cells in six-well culture vessels were transfected with 5 μg DNA using Xfect (Takara), and clones were selected by G418 (400–800 μg/mL) or puromycin (2–10 μg/mL).
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