HEK293 stable cells with tetracycline-inducible CIRP expression were generated using the Flp-In system. The CIRP sequence was cloned into a pTO destination vector (generated by Stephan Geley) and the plasmid was cotransfected with the pOG44 plasmid (Invitrogen) encoding the Flp recombinase into HEK293-Flp-In T-REx cells. Stable Flp-In expression cells were selected for antibiotic resistance.
For constitutive knockdown of CIRP, oligonucleotides (listed in Supplementary Table S1) were annealed and cloned into the BglII–HindIII site of the pENTR-THT III Gateway vector (62 (link)) and introduced into the lentiviral destination vector pHR-dest-SFFV-Puro by LR recombination. Lentiviral particles containing CIRP shRNA or control luciferase shRNA were generated in HEK293T packaging cells and used to infect target cells. Transduced cells were selected for puromycin resistance.