Extended Data Fig. 2: Library screen for NIK-stimulated genes.
From: A NIK–SIX signalling axis controls inflammation by targeted silencing of non-canonical NF-κB
a, Schematic of NIK-stimulated gene library design, cloning, and multidimensional flow cytometry-based high-throughput screen. NIK-stimulated genes were identified by RNA-seq. The cDNAs of 237 NIK-stimulated genes were individually cloned into the lentiviral vector pTRIP upstream of IRES-tagRFP (see Methods). Fibroblasts were transduced with lentivirus in a one-gene to one-well format and were then infected with GFP-expressing pathogens, including Lm, Sf, EAV, WNV, SINV, or PIV3 in independent experiments. The effect of a single gene expression on infection was quantified by flow cytometry. b, Relative expression levels of NIK-stimulated genes identified by RNA-seq. Fluc or NIK lentivirus was transduced into fibroblasts. Total RNA was isolated after 72 h and gene expression was determined by RNA sequencing. Graph shows expression of genes that were significantly stimulated (red, 237 genes) or downregulated (green, 84 genes) by NIK expression compared to Fluc control. Fold change of more than 2 (log2 ≥ 1) or less than 0.5 (log2 ≤ −1) and FDR < 0.05 (see Methods). Bars were ranked numerically from low to high (see Supplementary Table 1). The expression levels of SIX1 and SIX2 are indicated. Data are representative of two independent experiments. c, Efficiency of lentiviral expression of NIK-stimulated genes used in the high-throughput bacterial and viral screen. NIK-stimulated genes were transduced into fibroblasts in a ‘one-gene to one-well’ format. Transduction efficiency measured as per cent RFP+ cells was determined by flow cytometry and was ranked numerically from low to high (see Source Data, values are the average of two technical replicates). Twelve out of 237 genes were poorly transduced (less than 20% RFP+) and were excluded from subsequent analyses. d, Dot plots of S. flexneri, L. monocytogenes, EAV, WNV, SINV, and PIV3 infectivity in the presence of expressed NIK-stimulated genes (in c). Data were normalized to the average of each screen, indicated as the black dotted line. We chose to confirm hits in Fig. 1d on the basis of two criteria: (1) the effect of gene expression on inhibiting or enhancing pathogen infection by less than or greater than 50%; and (2) an adjusted z-score less than −2 or greater than 2 (see Supplementary Table 2). NIK-stimulated genes that reproducibly and significantly inhibited (green) or enhanced infection (red) by these criteria are indicated. The genes shown in black font are hits that were not reproduced in the confirmatory experiments (Fig. 1d). Data are mean ± s.d. from two (S. flexneri, L. monocytogenes) or one (EAV, WNV, SINV, and PIV3) independent experiments.
