Fig 2: Acquisition of stemness traits through concerted action of activated NF-κB and β-catenin. (a) Effect of SK3842 infection on undifferentiated cells: (i) Presence of 16S rRNA DNA in CD44high and CD44low cells after SK3842 re-infection. (ii) Changes in levels of indicated proteins in primary spheroids of Caco-2 cells following multiple SK3842 infections in SFM (adh=adherent). (b) Effect of SK3842 infection on differentiated cells: (i) mRNA expression of SC markers and (ii) Percentage of CD44high cells in differentiated Caco-2 cells following multiple SK3842 infections (post-diff=starting population). (c) Effect of NF-κB activation (IκBα shRNA) and inhibition (Bay11-7082) on (i) expression of SC markers and (ii) PARP-1 cleavage (5 days after final infection). (d) Effect of β-catenin activation (LiCl) and inhibition (JW67) on expression of SC markers. (e) Expression of SC markers in bacteria-cleared infected cells under spheroid-forming and adherent conditions. (f) Stemness-associated protein expression in uninfected cells treated with 30 and 70% sterile conditioned media from infected cell culture: (i) Protein levels in representative blot. (ii) Summary histogram of protein levels from independent blots. Data=Mean±S.D. from three experiments (*P<0.05, **P<0.01 and ***P<0.001)

Fig 3: Inhibition of autophagy increases TMQ0153-induced necroptosis.a Effect of autophagy inhibitors (40 nM baf-A1, 10 mM, 3-methyladenine (3-MA) and 75 μM chloroquine (CQ)) on death of K562 cells treated with 30 µM of TMQ0153 assessed by nuclear morphology analysis after 8 h of treatment. b-e K562 cells were transfected with specific small interfering (si)RNAs against beclin 1 [(SiBCN-1), 5 nM SiBCN-1_1 and 10 nM SiBCN-1_2] for 24 h. b Upper panel: effect of siRNA on beclin 1 protein expression level. After quantification of the bands, beclin 1 levels were normalized to β-actin. Lower panel: transfected cells were treated with 30 μM of TMQ0153 and a nuclear morphology analysis was carried out after 8 h of treatment. c-e Effect of siRNA on TMQ0153-induced autophagy quantified by flow cytometry after Cyto-ID staining (c); on caspase-3/7 activity (d), and PARP-1 cleavage using C2-10 antibody (e). Etoposide (Eto; 100 µM, 24 h) and EtOH (10 %, 2 h) were used as positive controls for apoptotic and necrotic PARP-1 cleavage (upper panel) and the corresponding densitometric analysis of necrotic cleavage (lower panel) is visualized. In western blot analyses, β-actin was used as a loading control. All pictures are representative of three independent experiments and data represent mean (±S.D.) of three independent experiments Statistical significance was assessed as *p < 0.05, **p < 0.01, ***p < 0.001 for the indicated comparisons. Two-way ANOVA (nuclear morphology analysis); post hoc; Sidak’s test. One-way ANOVA (Cyto-ID assay); post hoc; Dunnett’s test. One-way ANOVA (caspase 3/7 assay); post hoc; Tukey’s test. One-way ANOVA (western blot quantification); post hoc; Dunnett’s test.

Fig 4: Rtel1 Telomeric Dysfunction Is Rescued by Blocking Replication Fork Reversal, Related to Figure 5(A) Quantification of telomere length heterogeneity per metaphase 96 hours after Ad-GFP or Ad-Cre infection. Boxplots represent the quantification from at least 30 metaphases from a representative experiment (****p < 0.0001; two-way ANOVA).(B and D) Quantification of telomere loss (B), telomere fragility (C), and telomere length heterogeneity (D) per metaphase 96 hours after Ad-GFP or Ad-Cre infection. Boxplots represent the quantification from at least 30 metaphases from a representative experiment (****p < 0.0001; two-way ANOVA).(E) Western Blot analysis of the different genotypes to monitor loss of endogenous PARP1 96 hours after Cre infection, as well as total PARylated proteins.(F) Images show a representative metaphase telomere FISH of the indicated genotypes. Telomere loss, indicated with yellow arrows; telomere fragility, indicated with red arrows; telomere length heterogeneity, indicated with white arrows.(G) Quantification of telomere length heterogeneity per metaphase 96 hours after Ad-GFP or Ad-Cre infection. Boxplots represent the quantification from at least 30 metaphases from a representative experiment (****p < 0.0001; two-way ANOVA).(H) Western Blot analysis of the different genotypes to monitor loss of endogenous UBC13 (left) and ZRANB3 (right) 96 hours after Cre infection.(I and K) Quantification of telomere loss (I), telomere fragility (J), and telomere length heterogeneity (K) per metaphase 96 hours after Ad-GFP or Ad-Cre infection. Boxplots represent the quantification from at least 30 metaphases from a representative experiment (****p < 0.0001; two-way ANOVA).(L) Quantification of the interaction between TERT and TRF1 as determined by in situ PLA assay in the different indicated treatments. Data represents quantification from at least 150 nuclei from a representative experiment (****p < 0.0001; two-way ANOVA).(M) Analysis of telomerase activity determined by TRAP assay in the presence of BSA or recombinant RTEL1. Telomerase activity was measured relative to the control and normalized to the internal standard (IS).

Fig 5: CtBP loss triggers caspase 8-dependent apoptosis.a–e KURAMOCHI cells were infected with indicated shRNAs. CtBP RNAi efficiency was analyzed by western blotting (a). Viable cells were counted by Trypan blue exclusion assay. ***p < 0.001 as compared with shCtrl group (b). Four days post infection, cell apoptosis was analyzed by Annexin V/PI staining (left panel); percentage of Annexin V positive apoptotic cells is shown in the bar graph (right panel), ***p < 0.001 as compared with shCtrl group (c). Cleaved caspases and PARP1 were analyzed by western blotting (d) and caspase 8 and caspase 3/7 activities were determined by enzymatic assay (e), ***p < 0.001 as compared with shCtrl group. f KURAMOCHI cells, infected with lentiviral shRNA’s as indicated, were treated with vehicle or Z-IETD-FMK (20 µM) for 6 days. Viable cells were counted by Trypan blue assay at indicated time points, ***p < 0.001 as compared with shCtrl group. g, h KURAMOCHI cells were transfected with siRNAs followed by infection with shRNAs. Cleaved caspase 8 was examined by western blotting (g), and viable cells were counted at indicated time points by Trypan blue assay (h). ***p < 0.001 as compared with shCtrl group.