Puromycin

To establish stable 293T clones expressing EK or EK-X2, we transfected the cells with a pcDNA vector (Thermo Fisher) expressing the EK or EK-X2 cDNA together with a hygromycin-resistance gene. After selection with 150 μg/ml hygromycin B (Thermo Fisher), several independent clones expressing EK or EK-X2 were isolated and analyzed.
To establish stable A549 cells, we used a lentiviral vector (the pLVX series, TaKaRa) to express small hairpin RNAs (shRNAs) under the control of the U6 promoter (the target sequences knocked down are listed in Table 3), together with a puromycin-resistance gene or blasticidin-resistance gene under the control of the PGK promoter. The recombinant lentiviruses were constructed by simultaneously transfecting 293T cells with the pLVX vector and lentiviral plasmids expressing Gag/Pol, Rev, and VSVG (Naldini et al., 1996 (link)). After selection with 1.3 μg/ml puromycin (Nacalai Tesque, Inc., Kyoto, Japan), the puromycin-resistant A549 cells expressing RIG-I-targeting shRNA were pooled, and stored for analysis and subsequent experiments. The sh-RIG-I A549 cells were then infected with recombinant lentiviruses expressing shRNAs targeting different sites in the EK mRNA (sh-EK#1, sh-EK#2, and control shRNA), and selected with 180 μg/ml blasticidin S (Wako Pure Chemical Industries, Ltd, Osaka, Japan). The blasticidin-resistant cells were pooled and analyzed.

Plasmid DNA (1 μg) was transfected into suspended DU145, PC3 or 22Rv1 cells (1 × 10⁵ cells) using the NENO system (Invitrogen) (voltage; 1150, width; 30, pulse number; 2). Transfected DU145 cells were cultured with 1.0 μg/mL puromycin (Nacalai Tesque) or 2 mM G418 (Nacalai Tesque), while transfected PC3 and 22Rv1 cells were respectively cultured with 4.0 μg/mL and 0.5 μg/mL puromycin. Single colonies were selected and individually cultured. pX330 including the target gRNA sequence (1 μg) and pPGK-puro (0.5 μg) were transfected into suspended DU145 cells (1 × 10⁵) using the NENO system; the DU145 cells were then cultured with 1.0 μg/mL puromycin for 1 week and then cultured without puromycin. Single colonies were selected and individually cultured. Genotypes were identified by PCR, which was performed using Q5 high-fidelity DNA polymerase (New England Biolabs Inc. (NEB), MA, USA).

293T and HEK293 cells were maintained in low glucose Dulbecco’s modified Eagle’s medium (Nacalai Tesque) supplemented with 10% fetal bovine serum (Sigma-Aldrich), 100 units/mL penicillin and 100 μg/mL streptomycin (Nacalai Tesque). Cells were transfected with plasmid DNAs using polyethylenimine ‘MAX’ (Polysciences, Warrington, PA, USA) and then selected with Puromycin (Nacalai Tesque) for pCAGIPuro vectors to establish stable cell lines.