Fig. 4: TNF-induced TBK1 activation is impaired in Tank/Azi2^DKO macrophages.

a, b Immunoblot analysis of lysates of bone marrow-derived macrophages (BMDMs) isolated from WT or Tank/Azi2^DKO mice and stimulated with TNF (100 ng/ml) as indicated. A representative experiment (a) and quantification of three independent experiments using BMDMs independently isolated from 3 animal pairs. Data were normalized to the maximal response of WT cells, mean ± SEM (b). Statistical analysis was based on the AUC for each cell line, unpaired two-tailed t-test. c, d RNAseq analysis of WT and Tank/Azi2^DKO BMDMs stimulated for two hours with TNF (100 ng/ml). The BMDMs were independently isolated from 3 pairs of animals, and RNA was isolated in five separate experiments. Heatmap shows the expression of selected TNF-responsive genes (c). Gene set enrichment analysis (GSEA) shows the enrichment of 105 genes induced by TNF stimulation (log₂ fold change > 1 and padj < 0.01; samples from WT and Tank/Azi2^DKO BMDMs were analyzed together). The ranked gene list represents the contrast between unstimulated Tank/Azi2^DKO and WT BMDMs (blue) or stimulated Tank/Azi2^DKO and WT BMDMs (red). The p-value was estimated using an adaptive multi-level split Monte-Carlo scheme in the fgsea R package (d). e Analysis of cell death induced upon TNF (100 ng/ml) stimulation of WT or Tank/Azi2^DKO BMDMs. Induction of cell death was detected via the Incucyte imaging system. Mean + SEM from eight separate experiments using samples isolated from 4 animal pairs, two-way ANOVA with Tukey’s post-tests. n.s., not significant. #, nonspecific band.