Fig. 1: A haploid genetic screen reveals regulators of NOD2.
From: Phosphorylation of muramyl peptides by NAGK is required for NOD2 activation
a, CRISPR engineering of the IL1B locus, resulting in an endogenous in-frame fusion of IL-1B and mScarlet separated by a self-cleaving peptide (T2A). b, Flow cytometry analysis of IL-1B–mScarlet induction following L18-MDP stimulation of wild-type (WT) and NOD2-knockout (KO) clonal KBM-7-IL-1BmScarlet cells for 16 h. Data shown for one representative of three independent experiments, indicating gating strategy. AU, arbitrary units. c, Flow cytometry analysis of IL-1B–mScarlet induction in wild-type and NOD2-knockout clonal KBM-7-IL-1BmScarlet cells stimulated as indicated for 16 h. Data are mean ± s.e.m of n = 3 independent biological samples. Two-way ANOVA with Šídák’s multiple-comparisons test. Ctrl, control. d, Genetic screen showing positive and negative regulators of IL-1B–mScarlet in cells treated with L18-MDP for 16 h. For each gene (dots), the ratio of the mutation frequency in cells with high IL-1B–mScarlet expression versus those with low IL-1B–mScarlet expression is plotted against the combined number of unique mutations identified in both populations of cells. Genes enriched for mutations are shown in black (two-sided Fisher’s exact test, false discovery rate (FDR)-corrected P value (Padj) < 0.05). Genes encoding known components of the NOD2 pathway (orange dots) and the novel regulator NAGK (blue dot) are indicated. e, Model of the NOD2 signalling pathway. Products of genes identified in the screen are highlighted in orange; REL and SPI1 are not depicted. ****P < 0.0001.
