Supplementary Figure 1: Control experiments for Figure 1.

(a) Localization of SETX in untreated or PR8ΔNS1 virus infected A549 cells (4hours). Nuclear (DAPI) and Tubulin staining are shown. SETX antibody specificity was validated using human fibroblast cells proficient (WT) or deficient for SETX (SETX deficient). Shown are representative images from one of two independent experiments. Scale bars represent 50μm. (b) SETX co-elutes with high molecular weight complexes. Lysates from uninfected or PR8ΔNS1 infected A549 cells were subjected to glycerol gradient sedimentation and the presence of SETX, RNAPII or the Influenza A RNA polymerase PB2 subunit in sequential fractions revealed through immunoblotting. LARP7 is a control for elution in high molecular weight complexes. (c). Co-immunoprecipitation of RNAPII and SETX in the absence or presence of RNaseA. (d). Western blot analysis of SETX depletion in siSETX cells relative to siCtrl and untreated (No Si) condition prior to and after infection with PR8ΔNS1 virus. (e) The expression levels of 12 housekeeping genes were determined by microarray analysis of un-transfected (No Si), siSETX- or siCtrl- transfected cells under basal conditions (without infection). Results for individual probesets are shown for genes that are represented by multiple probesets on the microarray. A full list of genes and their expression in the experimental condition tested can also be found in Supplementary Table 1. (f) Western blot analysis of XRN2 depletion in siXRN2-treated cells relative to siCtrl and untreated (No Si) conditions prior to and after infection with PR8ΔNS1 virus. (g) Heatmap showing differentially expressed genes (Fold change relative to siCtrl >1.5, p < 0.01; ANOVA with post-hoc TUKEY test) in un-transfected cells (No si) or XRN2 siRNA (siXRN2) transfected cells as compared to control non-targeting siRNA transfected cells (siCtrl) at an early (4 hours) time point post-infection. (h). The expression levels of 12 housekeeping genes were determined by microarray analysis of un-transfected (No Si), siXRN2 or siCtrl transfected cells under basal conditions (without infection). Results for individual probesets are shown for genes that are represented by multiple probesets on the microarray. A full list of genes and their expression in the experimental condition tested can also be found in Supplementary Table 2. (i). Protein levels of SETX and phosphorylated IRF3 (pIRF3) were measured in siCtrl and siSETX cells left untreated or infected with PR8∆NS1 virus. Tubulin protein levels were used as a loading control. (j and k) Expression of virus-derived messenger RNA (j), and viral genomic RNA (k) from SETX siRNA transfected (siSETX, white bars), control siRNA transfected (siCtrl, Gray bars) and un-transfected (No si, black bars) cells infected for 4 hours with the PR8∆NS1 virus. Data shown are from three experiments (e-f, g-h and j-k; with the mean and s.d shown for j-k). A representative blot of two independent experiments (b and c) or blots of pooled extracts from two independent experiments are shown (d, f).