Fig 2: CRISPR-Cas9 mediated knockout of TNFR1 in HeLa cells resulted in abrogation of Eriodictyol mediated apoptosis. A. Western immunoblot-based screening and selection of TNFR1 negative clones from an array of clones selected by puromycin treatment (1mg/ml). B. Western immunoblot showing TNFR1 expression in HeLa (wild-type) versus TNFR1 KO-6 clone. C. TNFR1 KO-6 clone shows 4bp insertion (TATA) that results in non-functional protein production. Inserted bases highlighted. The boxed portion represents the targeted DNA sequence. D, E. Density plot and bar graph from FACs analysis showing significant abrogation of apoptosis in TNFR1 KO-6 clone compared to HeLa (wild-type) upon Eriodictyol treatment at 150 µM dose for 48 h. Data has been collected from three independent experiments and analyzed by Student's t-test (two-tailed).

Fig 3: IFN-? and TNF-a kill IECs through non-canonical TNFR1 signalling.A HT-29 cells were transfected with a nontargeting siRNA (siCtrl), siTNFR1, siTNFR2 or positive control siRNA’s (siJAK1, siJAK2), 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 and RT-qPCR validation of TNFR1/2 knockdown (right). B–D HT-29 cells were pretreated for 1 h with vehicle (DMSO), RIPK1 inhibitor (necrostatin-1s, 10 µM), RIPK3 inhibitor (GSK'872, 10 µM), MLKL inhibitor (NSA, 1 µM), dual JAK1/2 inhibitor (baricitinib, 10 µM) alone or in combination with a pan-caspase inhibitor (zVAD, 20 µM), followed by IFN-?/TNF-a (10 ng/ml each). B SYTOX Green/Annexin V signals and caspase-3 activity (heatmap) were recorded at indicated times. C Crystal violet staining and its absorbance quantification (A590 nm) at 72 h. D Relative viability measured at 72 h. E, F Primary human colonic organoids derived from CD colonic biopsies (n = 3 lines) were pretreated for 30 min with the indicated inhibitors (concentrations as in section B), followed by IFN-?/TNF-a (10 ng/ml each). E Relative viability and F SYTOX Green staining were assayed at 8 h after cytokine treatment. Data were mean ± SEM of n = 3 independent experiments. **p < 0.01 and ***p < 0.001 (two-way ANOVA with Tukey’s multiple comparisons test), ###p < 0.001 (unpaired t-test). NT non-treated, nec-1s necrostatin-1s, G'872 GSK'872, G + N GSK'872 + NSA, BARI baricitinib.

Fig 4: Etanercept reversed the effects of CSE on levels of inflammatory cytokines, TNFR1, p-p65/ p65 in HPAECs. (A and C) Levels of inflammatory cytokines TNF-a (A), IL-6 (B) and IL-8 (C) in HPAECs after CSE exposure and ETN treatment were quantified using enzyme-linked immunosorbent assay (ELISA). (D–F) Protein/ß-actin expressions of TNFR1, p65 and p-p65 in HPAECs after CSE exposure and ETN treatment were determined using Western blot. ß-Actin was used as the internal control. All experiments were performed in triplicate and experimental data were expressed as mean ± standard errors (SE). *P<0.05, vs control; #P<0.05, vs CSE.

Fig 5: The biological function of pharmacological APT2 inhibition in TNF signaling. The dashed black lines indicate wt apoptosis and internalization levels upon stimulation with TNF. Abbreviation: ML349 (349). All experiments show mean values (+/- SD) of 3–5 independent experiments. a TNF-R1 internalization in response to activation by TNF. Ctrl: TNF-R1 internalization increases upon 30 min incubation at 37 °C in the presence of TNF. Incubation with ML349 (I: 50 µM, II: 25 µM, III: 12.5 µM, IV: 6.35 µM) reduced internalization. b Apoptosis induction analyzed by quantification of nuclear DNA fragmentation. 20 h TNF treatment increased apoptotic cells. Incubation with ML349 (25 µM) increased apoptosis rate. Higher concentrations of ML349 resulted in excess apoptosis without TNF. c Apoptosis induction analyzed by WB. Panel one: PARP1 cleavage. Panel two: active caspase-3. Panel three: actin as loading control. d Ceramide production after 10 min stimulation with TNF. TNF induces ceramide formation, which is increased upon APT2 inhibition by ML349. nSMase inhibition by GW4869 (10 µM) protects from ceramide production. e Caspase-3 activity was monitored up to 70 min upon addition of 100 ng/ml TNF, 25 µM ML349, 2 µM GW4869. One representative experiment of three measurements in duplicates is shown. f Degradation of I?B was analyzed upon stimulation with TNF by WB. Actin serves loading control. One representative experiment is shown