a, b, Experiments were performed to exclude the possibility that the observed CD40L-induced antibacterial function was specific to a particular cell type or protocol of cytokine induction. We reconstituted the CD40L signalling pathway in HEK293 cells. These cells do not express CD40, the endogenous receptor for CD40L (a, left). HEK293 cells are also unable to be stimulated by CD40L (a, right). However, overexpression of CD40 strongly induced NF-κB pathway activation (a, right). Expression of CD40 restricted both L. monocytogenes and S. flexneri infection (b) to levels similar to those observed in CD40L treated U-2 OS cells (compare with Fig. 1b). The NF-κB reporter activity assay (a, right) was performed by co-transfecting empty vector (EV), CD40L or CD40 with 5 × κB-LUC reporter gene into HEK293T cells. Luciferase activity was measured after 48 h and normalized to EV. Data are mean ± s.d. from six independent experiments. Experiment and quantification of b are presented as in Fig. 1b. Data are mean ± s.d. from six independent experiments. c, d, Confirmation of genetic knockout of the MAP3K14 (NIK) and MAP3K7 (TAK1) genes in STAT1−/− human fibroblasts. c, Schematic representation of In/Del base pairing and the sgRNA targets locus of exon 1 in NIK and TAK1. NIK−/− contains deletion of (−)7 bp, insertion of (+)G, and +CTCAC alleles (top). TAK1−/− contains +AT alleles, −2 bp, and −409 bp (bottom). d, Endogenous NIK and TAK1 expression in parental, NIK−/− and TAK1−/− cells. NIK is constitutively degraded by the cIAP–TRAF2/3 E3-ligase complex in quiescent cells2. Wild-type, NIK−/−, and TAK1−/− fibroblasts were treated with 2.5 μM BV6 for 14 h and then probed with indicated antibodies using western blotting. e, NIK is necessary for restricting L. monocytogenes infection. Fibroblasts with the indicated genetic background were treated with vehicle control (DMSO) or 2.5 μM BV6 for 14 h and then infected with LmGFP. Percentage bacterial infection was normalized to wild-type untreated control. Black asterisks denote the difference between wild-type and indicated cell lines; red denote the difference between DMSO and BV6 treatment. NIK−/− cells exhibited much higher L. monocytogenes infection than either wild-type or TAK−/− cells, consistent with the role of NIK in preventing infection after cellular stimulation. However, BV6 treatment—which suppressed L. monocytogenes infection of wild-type cells—had no effect on NIK−/− cells, further indicating that NIK activation is necessary for the antibacterial response. Data are mean ± s.d. from nine independent experiments. f, NIK kinase activity is required for its antibacterial function. Fluc, wild-type NIK or NIK-kinase dead mutant (NIKK429/430A referred to as NIKKD) lentivirus was transduced into fibroblasts or U-2 OS cells as indicated. Cells were then challenged with SfGFP. Quantification of bacterial infection is as in Fig. 1b. Data are mean ± s.d. from four independent experiments. g, NF-κB gene expression induced by NIK is kinase dependent. EV, NIK or NIKKD was co-transfected with 5 × κB-LUC into HEK293T cells. NF-κB activity was measured after 48 h and normalized to EV. Data are mean ± s.d. from four independent experiments. h, Expression of NIK, but not TAK1, inhibits infection with L. monocytogenes and S. flexneri. Wild-type U-2 OS cells were transduced with combinational FlucRFP/FlucBFP, NIKRFP/FlucBFP, or TAK1RFP/TAB1BFP lentivirus. Cells were then challenged with LmGFP or Sf GFP. Infection efficiency was quantified by flow cytometry, gating GFP+ cells in both RFP+ and BFP+ cell populations. The relative percentage of pathogen infection was normalized to Fluc control. Data are mean ± s.d. from eight independent experiments. i, NIK protein expression levels corresponding to experiments in Fig. 1c. Fluc- and NIK-transduced cells were lysed and probed with anti-NIK antibody. j, Previous studies have suggested that ectopic expression of NIK is cytotoxic in A549 cells44. To test whether ectopic expression of NIK is cytotoxic in fibroblasts, we transduced fibroblasts with indicated lentivirus and measured cell viability after 72 h by measuring ATP. Data are mean ± s.d. from six independent experiments. P values were measured using one-way ANOVA (GraphPad); ***P < 0.001, ****P < 0.0001; ns, no significant difference. The same statistics were used in later figures unless otherwise stated. Western blot data are representative of three independent experiments. For gel source data, see Supplementary Fig. 1. Source data