Fig 2: Effect of 5-aza-dc treatment on mRNA, protein expression and promoter methylation of CASP8 and maspin. Cells were treated with 5 µM 5-aza-dc for 3 days. (A) RNA were isolated from the treated and untreated cells and performed for RT-PCR with CASP8 primers. ß-actin was used as loading control. (B) Total protein was isolated after cells were treated or untreated with 5-aza-dc. Western blot analysis was performed with antibodies specific for full length caspase-8, and ß-actin was used as loading control. (C) DNA were isolated and modified by bisulfite treatment. MSP was performed with methylated and unmethylated CASP8 PCR primers as described in Methods. The bands detected by methylated primer represented methylated maspin (M), and the bands detected by unmethylated primer represented unmethylated maspin (UM). (D) The DNA from 5-aza-dc treated and untreated cells were modified by bisulfite treatment and amplified with CASP8 primer designed according to the bisulfite modified sequence of CpG sites in CASP8 promoter as described in Methods. The amplified PCR products were purified and sequenced for confirmation the CpG sites methylation. The dark dots represented methylated CpG sites in the promoter, and open dots indicated unmethylated CpG sites. (E) RNA from 5-aza-dc treated and untreated cells were used to perform RT and real-time PCR with maspin primers. The relative level of maspin gene was normalized to 18 s as described in Methods. Each bar represented the mean of three independent amplifications with standard deviation (SD). (F) DNA from cells treated and untreated with 5-aza-dc were modified by bisulfite treatment, and MSP was performed with methylated and unmethylated maspin PCR primers as described in Methods. The bands detected by methylated primer represented methylated maspin (M), and the bands detected by unmethylated primer represented unmethylated maspin (UM).

Fig 3: Activation of the CASP8-JAK1/2-STAT1 cell death module in human colonic biopsies and organoids.A–C Colonic biopsies isolated from healthy (HL) individuals (n = 10) or patients with inactive/non-inflamed (n = 11) and active/inflamed (n = 11) Crohn’s disease (CD) were incubated ex vivo with anti-CD3/28 (5 µg/ml each) for 18 h. Relative mRNA expression of A IFNG, TNF and IL2, and B JAK1, JAK2, STAT1 and CASP8 measured by RT-qPCR. C Correlation between relative mRNA levels of the indicated genes or the JAK-STAT score in resting (left) and anti-CD3/28-stimulated biopsies (right). D Relative viability of primary human colonic organoids derived from HL (n = 3 lines) or CD (n = 3 lines) colonic biopsies, and stimulated with IFN-? and/or TNF-a (10 ng/ml each) for the indicated times. Coefficient of cytokine interaction (CCI) calculated per time-point (right). E–G Primary human colonic organoids derived from CD colonic biopsies (n = 3 lines) were pretreated for 1 h with vehicle (DMSO), JAK1-selective compound (upadacitinib and filgotinib), JAK1/2 inhibitor (baricitinib) or pan-JAK inhibitor (tofacitinib) at 0.1, 1 or 10 µM, followed by IFN-? and/or TNF-a (10 ng/ml each) for the indicated times. E STAT1 activity was measured by HTRF assay at 2 h (top) and relative viability at 8 h (bottom) of cytokine treatment. F SYTOX Green staining of a select organoid line at 8 h of cytokine treatment. G Pearson correlation between relative viability and STAT1 activity. Data were mean ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.001 (two-way, repeated-measures ANOVA with Bonferroni’s multiple comparisons test), #p < 0.05, ##p < 0.01 and ###p < 0.001 (two-way ANOVA with Tukey’s multiple comparisons test), /p < 0.05, ///p < 0.001 (one-way ANOVA with Dunnett’s multiple comparisons test vs. DMSO plus IFN-?/TNF-a). NT non-treated, JAKinib JAK inhibitor, UPA upadacitinib, BARI baricitinib, TOFA tofacitinib, FILGO filgotinib, org. organoids, CCI coefficient of cytokine interaction.

Fig 4: Caspase-8 nonenzymatic function is required for IEC killing by IFN-?/TNF-a.A HT-29 cells were pretreated for 1 h with vehicle (DMSO), caspase-8 inhibitor (Z-IETD, 10 µM), caspase-10 inhibitor (Z-AEVD, 10 µM) or zVAD (20 µM), followed by IFN-?/TNF-a (10 ng/ml each). SYTOX Green/Annexin V signals and caspase-3 activity (heatmap) were recorded at indicated times, and relative viability was measured at 72 h (inner graph). B HT-29 cells were transfected with a nontargeting siRNA (siCtrl), siCASP8, siCASP10 or a positive control siRNA (siSTAT1), followed by IFN-?/TNF-a (10 ng/ml each) for 72 h. SYTOX Green/Annexin V signals and caspase-3 activity (heatmap) were recorded at indicated times, and relative viability was measured at 72 h (inner graph). For both targets, siRNA’s 'a' were used. C Parental, CASP8 or CASP10 knockout HT-29 cells were treated with IFN-?/TNF-a (10 ng/ml each). SYTOX Green/Annexin V signals and caspase-3 activity (heatmap) were recorded at indicated times, and relative viability was measured at 72 h (inner graph). Western blot validation of CASP8/10 knockout (bottom). D Parental or CASP8 knockout DLD-1 cells were pretreated for 1 h with vehicle (DMSO), caspase-8 inhibitor (Z-IETD, 10 µM) or zVAD (20 µM), followed by IFN-?/TNF-a (10 ng/ml each). SYTOX Green/Annexin V signals and caspase-3 activity (heatmap) were recorded at indicated times, and relative viability was measured at 72 h (inner graph). Western blot validation of CASP8 knockout (bottom). E STAT1 activity was measured by HTRF assay in parental and CASP8 knockout HT-29 treated with IFN-? and/or TNF-a (10 ng/ml each) for indicated times. F Western blot analysis of JAK1/2, STAT1 (total, Y701- and S727-phosphorylated) and CASP8 expression in parental and CASP8 knockout HT-29 treated with IFN-?/TNF-a (10 ng/ml each) for 16 h. G STAT1 activity at 16 h measured by HTRF assay (left), and crystal violet staining at 72 h (right) in parental and CASP8 knockout HT-29 pretreated for 1 h with DMSO or caspase-8 inhibitor (Z-IETD, 10 µM), followed by IFN-?/TNF-a (10 ng/ml each). Data were mean ± SEM of n = 3 independent experiments. ***p < 0.001 (two-way ANOVA with Tukey’s multiple comparisons test), +p < 0.05, ++p < 0.01 and +++p < 0.001 (two-way ANOVA with Bonferroni’s multiple comparisons test vs. time point-matched parental HT-29 cells), ###p < 0.001 (unpaired t-test). NT non-treated, C8-i/CASP8-i caspase-8 inhibitor, CASP10-i caspase-10 inhibitor, KO knockout, I + T IFN + TNF.

Fig 5: Evaluation of the relationship between Draxin and tumorigenesis. (A) Western blot analysis of Draxin knockdown in human lung adenocarcinoma cells (H358). Three biological replicates were analyzed. Representative examples of no treatment (NT), transfected control siRNA (Si-cont.), and Draxin siRNA (Si-Draxin) are shown. Expression of phosphorylated-histone H3 (ser10) and CyclinD1 as mitotic markers, antibodies against Caspase3 (CASP3), Cleaved Caspase3 (cCASP3), Caspase8 (CASP8), Caspase9 (CASP9), B-cell lymphoma 2 (Bcl-2), and phosphorylated-Bcl-2 (pBcl-2) as apoptosis-related markers, and antibodies against AKT and phosphorylated-AKT (pAKT) as markers of cell signaling activity. Draxin knockdown was successfully performed in Si- Draxin cells. All markers showed similar expression patterns between NT and Si-cont. Draxin siRNA induced the suppression of CASP3, CASP8 and CASP9. In addition, Draxin siRNA elevated of pHH3 levels. ß-actin served as an internal control. (B) Ki-67 (MIB-1) index analysis of Draxin knockdown in human lung adenocarcinoma cell (H358). Upper panel, transfected control siRNA (control) where a moderate number of adenocarcinoma cells were positive for Ki-67. Lower panel, Draxin siRNA (Si-Draxin) where most adenocarcinoma cells were positive for Ki-67. Bar = 100 µm. (C) The average Ki-67 index in transfected control siRNA cells was 41.9%. The average Ki-67 index in Draxin siRNA cells was 63.6%. * p < 0.01.