Extended Data Fig. 6: ZBP1(S)-mediated cell death does not involve PANoptosis.
From: Telomere-to-mitochondria signalling by ZBP1 mediates replicative crisis
a, Immunoblotting of pre-crisis (PD97) IMR90E6E7 expressing empty vector or ZBP1(S)-Flag at day 10 post-transduction. Pre-crisis IMR90E6E7 treated with staurosporine (1 μM) serve as positive controls. GAPDH loading control. Two independent experiments were performed. b, Immunoblotting of pre-crisis (PD97) IMR90E6E7 expressing empty vector or ZBP1(S)-Flag at day 10 post-transduction. HT-29 cells treated with TBZ (TNF (1000 U ml−1) + BV6 (1 μM) + z-VAD-FMK (20 μM)) serve as positive controls. GAPDH loading control. Two independent experiments were performed. c, Immunoblotting of pre-crisis (PD97) IMR90E6E7 expressing empty vector or ZBP1(S)-Flag at day 10 post-transduction. THP-1 cells primed with LPS (1 µg ml−1) for 4 h prior to treatment with nigericin (10 µM) for 15 and 30 min serve as positive controls. GAPDH loading control. Two independent experiments were performed. d, Immunoblotting of pre-crisis (PD97) IMR90E6E7 expressing empty vector or ZBP1(S)-Flag at day 10 post-transduction. GAPDH loading control. Two independent experiments were performed. e, Scatter plot with bars showing the percentage of dead pre-crisis (PD97) IMR90E6E7 expressing either empty vector or ZBP1(S)-Flag at day 10 post-transduction. ZBP1(S)-Flag expressing cells were transfected with siRNAs at day 8 post-transduction and incubated with Cytotox green for 24 h before imaging. Dead cells are labelled with Cytotox green and nuclei with Hoechst. Bars represent mean ± s.d. from biological replicates. n: number of biological replicates. One-way ANOVA, ns: not significant, p** < 0.01, *** p < 0.001. Three independent experiments were performed. f, Left: Representative fluorescence microscopy images of Metaphase-TIF assays stained with DAPI (blue), telomere FISH (green) and γH2AX immunofluorescence (red) performed on growing (PD35) IMR90E6E7 expressing non-targeting control shRNA or shRNA against TRF2. Mock represents non-transduced cells. Experiments were performed at the indicated days post-shRNA transduction. Scale bar 10 μm. Three independent experiments were performed. Right: Representative fluorescence microscopy images of cytogenetic preparations stained with DAPI (red) and telomere FISH (green) performed on growing (PD35) IMR90E6E7 expressing non-targeting control shRNA or shRNA against TRF2. Mock represents non-transduced cells. Experiments were performed at the indicated days post-shRNA transduction. Scale bar 10 μm. Three independent experiments were performed. g, Box and whisker plots showing the number of telomeric γH2AX foci / metaphase (left) and number of fused chromosomes / metaphase (right) at the indicated days post-shRNA transduction. Centre line: median; box limits: 1st and 3rd quartiles; whiskers: 10th and 90th percentiles. n: number of metaphases analysed. One-way ANOVA, ns: not significant, *** p < 0.001. Three independent experiments were performed. i, Experimental timeline of Fig. 2a. Growing (PD35) IMR906E7 were first transduced with non-targeting control shRNA or shRNA against TRF2, selected, and transfected with either of two individuals siZBP1 or non-targeting control siRNA at day 4 post-shRNA transduction. Protein extracts and metaphase spreads were prepared at days 6,9, and 12 post-shRNA transduction. j, Box and whisker plots showing the number of fused chromosomes / metaphase at day 6 post-shRNA transduction. Centre line: median; box limits: 1st and 3rd quartiles; whiskers: 10th and 90th percentiles. n: number of metaphases analysed. One-way ANOVA, ns: not significant. Three independent experiments were performed. Abbreviations: KO: knock-out; stauro: staurosporine; TBZ: TNF-BV6-z-VAD-FMK; LPS: lipopolysaccharide; Cl.: cleaved; Casp: caspase; NT: non-treated; TIF: telomere-dysfunction-induced foci; FISH: fluorescence in situ hybridization; CTR: control; PDs: population doublings.
