Fig 2: Telomere dysfunction is required for ZBP1-dependent IFN signalling.a, Immunoblot analysis of growing (PD35) IMR90E6E7 fibroblasts expressing non-targeting control short hairpin RNA (shRNA) or shRNA against TRF2 (shTRF2). shTRF2 cells were transfected with either two individual siRNAs against ZBP1 (siZBP1) or non-targeting control siRNA at day 4 after shRNA transduction. Protein extracts were collected at days 6, 9 and 12 as shown in the timeline of the experiment in Extended Data Fig. 6i. Mock represents non-transduced cells. GAPDH was the loading control. Two independent experiments were performed. p, phosphorylated. b, RT–qPCR analysis of ISGs in growing (PD35) IMR90E6E7 fibroblasts expressing non-targeting control shRNA or shRNA against TRF2. shTRF2 cells were transfected with either two individual siRNAs against ZBP1 or non-targeting control siRNA (siCtrl) at day 4 after shRNA transduction. RNA extracts were collected at day 12. Expression levels were normalized to control cells with non-targeting shRNA. Data are mean ± s.d. of technical replicates. n values indicate the number of technical replicates. Statistical analysis was performed using one-way ANOVA; ***P < 0.001. Three independent experiments were performed. c, Immunoblot analysis of IMR90E6E7 fibroblasts expressing either empty vector or hTERT at the indicated PDs. Growth curves and telomere restriction fragment (TRF) analysis are shown in Extended Data Fig. 7a,b. Cells stimulated with 2 µg ml-1 of poly(deoxyadenylic-deoxythymidylic) (poly(dA:dT)) or poly(inosinic:cytidylic) (poly(I:C)) for 24 h were used as positive controls. GAPDH was used as the loading control. Two independent experiments were performed. d, Immunoblot analysis of growing (PD42), pre-crisis (PD100) and telomerase-positive (PD105) IMR90E6E7 fibroblasts expressing ZBP1(L) or ZBP1(S). Flag tag was added to either the N terminus or the C terminus. GAPDH was the loading control. Two independent experiments were performed. Lipo, lipofectamine.Source data

Fig 3: Sensing of TERRA by ZBP1 triggers an IFN response.a, Line chart showing growth curve analysis of IMR90E6E7 expressing either WT ZBP1(S)-Flag or ZBP1(S)-Flag lacking Za2 with either TRF1(?N) or VP16-TRF1(?N). Fresh media with doxycycline (1 µg ml-1) was added every 2 days. Two independent experiments were performed. b, Scatter plots showing RT-qPCR analysis of ISG levels in cells as in a. Expression levels were normalized to control cells expressing either WT ZBP1(S)-Flag or ZBP1(S)-Flag lacking Za2 with TRF1(?N). RNA extracts were collected at day 20 of the experiment. Bars represent mean ± s.d. from technical replicates. n: number of technical replicates. One-way ANOVA, ns: not significant, *** p < 0.001. Two independent experiments were performed. c, Line chart showing growth curve analysis of IMR90E6E7 expressing either WT ZBP1(S)-Flag or ZBP1(S)-Flag containing point mutations in Za2, with either TRF1(?N) or VP16-TRF1(?N). Fresh media including 1 µg ml-1 of doxycycline was added every 2 days. Two independent experiments were performed. d, Scatter plots showing RT-qPCR analysis of ISG levels in cells as in c. Expression levels were normalized to control cells expressing either WT ZBP1(S)-Flag or ZBP1(S)-Flag containing point mutations in Za2 with TRF1(?N). RNA extracts were collected at day 30 of the experiment. Bars represent mean ± s.d. from technical replicates. n: number of technical replicates. One-way ANOVA, ns: not significant, *** p < 0.001. Two independent experiments were performed. e, Experimental timeline. Growing (PD35) IMR90E6E7 expressing WT ZBP1(S)-Flag were treated with doxycycline (1 µg ml-1) prior to four sequential siRNA transfections. Two individual siRNAs targeting TERRA were used. Samples were collected at days 2,4,6, and 8 post-doxycycline. f, RNA-dot blot performed on RNA isolated from total cell lysates at day 8 post-doxycycline as shown in the timeline of the experiment, using 32P-dCTP-labelled probes targeting either TERRA or GAPDH transcripts. RNase A treatment was used to assess possible DNA contamination. GAPDH loading control. Three independent experiments were performed. g, Western blotting performed on protein extracts collected at days 2,4,6, and 8 post-doxycycline treatment as shown in e. GAPDH loading control. Two independent experiments were performed. Abbreviations: ZBP1(S): short isoform; WT: wild-type; CTR: control; PDs: population doublings. Source data

Fig 4: ZBP1-dependent signalling is mediated by MAVS.a, Growing and crisis IMR90E6E7 and WI38SV40LT fibroblasts, and crisis IMR90E6E7 and WI38SV40LT fibroblasts expressing WT ZBP1(S)–Flag or ZBP1(S)–Flag containing mutations in Za2, RHIM1 or RHIM2, immunostained against endogenous ZBP1 or Flag (left). Scale bar, 10 µm. Three independent experiments were performed. Right, box and whisker plots of endogenous ZBP1 filaments. The median (centre line), first and third quartiles (box limits) and 10th and 90th percentiles (whiskers) are shown. n values indicate the cell number. Statistical analysis was performed using one-way ANOVA; **P < 0.01; ***P < 0.001. Three independent experiments were performed. The white boxes indicate higher-magnification images. b, Immunoblot analysis of growing and pre-crisis IMR90E6E7 fibroblasts expressing empty vector, WT ZBP1(S)–Flag or ZBP1(S)–Flag containing domain point mutations. GAPDH was used as the loading control. Two independent experiments were performed. c, RT–qPCR analysis of ISGs in growing and pre-crisis IMR90E6E7 fibroblasts. Expression levels were normalized to growing cells expressing the empty vector. Data are mean ± s.d. of 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. d, Growing IMR90E6E7 fibroblasts expressing Flag–ZBP1(S) or Flag–ZBP1(S)–FIS1 immunostained for Flag and MAVS. Scale bar, 10 µm. Three independent experiments were performed. e, RT–qPCR analysis of ISGs. RNA extracts were collected 10 days after transduction. Expression levels were normalized to 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. ***P < 0.001. Three independent experiments were performed. f, Immunoblot analysis of growing IMR90E6E7 fibroblasts as described in e. GAPDH was used as the loading control. Two independent experiments were performed. g, RT–qPCR analysis of ISGs in growing (PD66) IMR90E6E7 fibroblasts. Cells were transfected with siMAVS or non-targeting control siRNA at day 10 after transduction. RNA extracts were collected at day 14. Expression levels were normalized to empty vector siCtrl cells. 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.Source data

Fig 5: Assembly of ZBP1 filaments at mitochondria does not require sensing of mtDNA or mtRNA.a, Upper panel: Representative confocal images of growing (PD50) IMR90E6E7 expressing either WT ZBP1(S)-Flag or ZBP1(S)-Flag lacking Za2 with either TRF1(?N) or VP16-TRF1(?N). Interphase nuclei were stained with DAPI (blue), telomere FISH (green), and ?H2AX immunofluorescence (red). Scale bar 5 µm. Middle panel: Representative fluorescence microscopy images of Metaphase-TIF assays stained with DAPI (blue), telomere FISH (green) and ?H2AX immunofluorescence (red). Scale bar 10 µm. Lower panel: Representative fluorescence microscopy images of cytogenetic preparations stained with DAPI (red) and telomere FISH (green). Scale bar 10 µm. Three independent experiments were performed at day 8 post-doxycycline treatment (1 µg ml-1). b, Immunoblotting of cells as in a either untreated or at days 4 and 8 post-doxycycline treatment (1 µg ml-1). Cells treated with 1 µM camptothecin (CPT) for 6 h serve as a positive control. GAPDH loading control. Two independent experiments were performed. c, Left: Representative confocal microscopy images of growing and crisis IMR90E6E7 co-immunostained for endogenous ZBP1 and mitochondrial markers TOM20, TFAM, and MitoTracker at the indicated PDs. Scale bar 10 µm. Right: Scatter plots showing the number of ZBP1 filaments colocalizing with either TOM20, TFAM, or MitoTracker in growing and crisis IMR90E6E7 and WI38SV40LT at the indicated PDs. Centre line: mean. n: number of cells analysed. One-way ANOVA, *** p < 0.001. Three independent experiments were performed. d, Left: Representative confocal microscopy images of crisis (PD100) IMR90E6E7 immunostained with antibodies against endogenous ZBP1, dsDNA, and dsRNA. Cells were incubated with 5 µM of 2’,3’ dideoxycytidine (ddC) for 14 days. Non-treated cells serve as controls. Scale bar 10 µm. Three independent experiments were performed. Right: Scatter plots showing the signal intensity of dsDNA (left), the signal intensity of dsRNA (middle), and the number of ZBP1 filaments (right). Centre line: mean. n: number of cells analysed. Three independent experiments were performed. Two-tailed Student’s t-test, ns: not significant, *** p < 0.001. Three independent experiments were performed. e, Left: Representative confocal microscopy images of growing (PD100) IMR90E6E7 transfected with either non-targeting control siRNA (siCTR), siSUV3, or PNPase, and co-immunostained for endogenous ZBP1 and double-stranded RNA (dsRNA). Experiment was performed at day 7 post-siRNA transfection. Scale bar 10 µm. Three independent experiments were performed. Right: Scatter plots showing the signal intensity of dsRNA (left) and the number of ZBP1 filaments (right). Centre line: mean. n: number of cells analysed. Two-tailed Student’s t-test, ns: not significant, *** p < 0.001. Three independent experiments were performed. Abbreviations: ddC: dideoxycytidine; dsDNA: double-stranded DNA; dsRNA: double-stranded RNA; Mito: mitochondria; WT: wild-type; CTR: control; TIF: telomere-dysfunction-induced foci; FISH: fluorescence in situ hybridization; CPT: camptothecin; WT: wild-type; PDs: population doublings. Source data