Construction of stable cell lines expressing mutant versions of FICD

The effect of FICD overexpression was analyzed in mammalian cell clones carrying stable transgenes that encode doxycycline-inducible mutant versions of FICD. To generate stable cell lines, plasmid DNA encoding FICD^E234G (UK1440) and FICD^E234G-H363A (UK1446) were introduced into the Flp-In T-REx 293 cell line (Invitrogen) as described by the manufacturer's protocol. Briefly, HindIII-XhoI DNA fragments containing the coding sequences for FICD^E234G and FICD^E234G-H363A were sub-cloned into pcDNA5/FRT/TO (Invitrogen). The resulting expression plasmids were individually co-transfected along with the Flp-recombinase expression vector pOG44 (Invitrogen) using Lipofectamine 2000 (Invitrogen) into Flp-In T-REx 293 cells. Isogenic clones expressing the transgene under a doxycycline-inducible promoter were selected for resistance to blasticidin (3 µg/ml; Thermo Fisher, UK) and hygromycin (250 µg/ml; Invitrogen) and sensitivity to zeocin (50 µg/ml; Invitrogen).