Fig. 1: ZBP1 mediates an innate immune response during crisis.
From: Telomere-to-mitochondria signalling by ZBP1 mediates replicative crisis
a, Pre-crisis IMR90E6E7 fibroblasts were transduced with the Brunello library (four sgRNAs per gene), genomic DNA was collected at days 0 and 15, and gRNA changes were measured (left). Right, enrichments. Two technical replicates. Data represent the log2-transformed fold change in read counts before and after enrichment (PinAPL-Py). gRNAs with a log2-transformed fold change of >2 are shown (Supplementary Information). Two independent experiments were performed. b, RNA-seq analysis of ISGs of growing (PD61), crisis (PD105) and pre-crisis (PD100) IMR90E6E7 fibroblasts that were transfected with two individual short interfering RNAs (siRNAs) against ZBP1 (siZBP1) or control (siCtrl). Significantly upregulated (crisis versus growing) and downregulated ISGs (siZBP1 versus siCtrl) with a fold change of >1.5 and a false-discovery rate (FDR)-adjusted P < 0.05 are shown (the complete list of ISGs used is from ref. 51). One experiment was performed. c, Representative IncuCyte images of pre-crisis (PD94) ZBP1-knockout IMR90E6E7 fibroblasts reconstituted with either WT ZBP1(S)–Flag or ZBP1(S)–Flag containing point mutations in Zα2, after 12 h of incubation (left). Dead cells are labelled with Cytotox green and nuclei are labelled with NucLight red. Scale bar, 150 μm. Three independent experiments were performed. Right, IncuCyte analysis of cell death of pre-crisis (PD94) ZBP1-knockout IMR90E6E7 fibroblasts reconstituted with either WT ZBP1(S)–Flag or ZBP1(S)–Flag containing point mutations in the Zα2, RHIM1 or RHIM2 domains, measured in real time by Cytotox green. Data are mean ± s.e.m. from technical replicates. Statistical analysis was performed using one-way analysis of variance (ANOVA); ***P < 0.001. Three independent experiments were performed. d, RT–qPCR analysis of ISGs. Expression levels were normalized to control cells with the empty vector. Data are mean ± s.d. from technical replicates. n values indicate the number of technical replicates. Statistical analysis was performed using one-way ANOVA; NS, not significant; ***P < 0.001. Three independent experiments were performed.
