Extended Data Fig. 1: siRNA knockdown of IFITs in A549 cells and expression of IFIT2 and IFIT3 in FlpIn lines, related to Fig. 1.
(a-c) A549 cells were treated with a non-targeting siRNA as a negative control or siRNA targeting human (a) IFIT1, (b) IFIT2, or (c) IFIT3. Twenty-four hours post-treatment, cells were harvested and subsequently processed and analyzed by western blotting. Representative images shown; 2 independent experiments. For downstream infection experiments, siRNA IFIT1-c and siRNA IFIT3-a were used. (d) We generated inducible Flp-In T-REx HEK293 cell lines co-expressing mouse IFIT2 and IFIT3. Cells were induced with 500 ng/mL DOX for 24 h before harvestting cell pellets for analysis by western blot. Representative images shown; 2 independent experiments. For all associated downstream experiments, Flp-In line b was used. (e) HEK293T were transfected with the indicated epitope-tagged plasmids expressing IFIT proteins from human (Hs) or mouse (Mm) and subjected to immunoprecipation with anti-HA conjugated beads to pull down HA-tagged human or mouse IFIT3. With pulldown of mouse IFIT3, we observe strong pulldown of mouse IFIT2, but no endogenous or overexpressed human IFIT1, which is consistent with previous literature26. In contrast, immunoprecipitation of human IFIT3 pulls down human IFIT2, and endogenous and overexpressed human IFIT1. Representative images shown; 2 independent experiments. (f) Inducible Flp-In T-REx HEK293 cells co-expressing mouse IFIT2 and IFIT3 were treated with a non-targeting control siRNA (NC, black circles) as a negative control or siRNA targeting IFIT1 (light grey squares). In indicated conditions, cells were mock treated or treated with doxycycline (500 ngμl−1) for 24 h and then infected with VSV-GFP (0.01 MOI), and supernatant was collected at 16 hpi for titration (n = 4 biological replicates). Statistical analyses: Data are represented as mean ± SEM. Ordinary two-way ANOVA with Tukey’s post-test and a single pooled variance. **** = p < 0.0001, ns = not significant.
