Generation of cell lines with stable knockdown or ectopic expression

iBMDM cells were transduced with lentiviruses for stable knockdown or ectopic expression. We generated lentiviruses by transfection of the pLKO.1 and pINDUCER20 constructs described above, together with: (i) an expression vector for the VSV-G envelope protein (pCMV-VSV-G, Addgene plasmid no. 8454); (ii) an expression vector for GAG-Pol-Rev (psPAX2, Addgene plasmid no. 12260); and (iii) a vector to aid with translation initiation (pAdVAntage, Promega) into 293 T cells using Lipofectamine 3000 Reagent (Invitrogen, L3000015) according to the manufacturer’s protocol. The resulting viruses were collected in the culture medium, concentrated by using a Lenti-X concentrator (Clontech, 631231), and used to infect iBMDM cells seeded at a density of 1 × 10⁶. Stably transduced cells were selected with puromycin (Sigma, P9620; 2.5 μg/mL) or G418 sulfate (Sigma, A1720; 1 mg/mL) in cell culture medium.