Fig 2: Caspase-9 cleavage was impaired under strong oxidative stress.A Caspase-3 activities in WT, caspase-3 KO (casp3), caspase-8 KO (casp8), and caspase-9 KO (casp9) HeLa cells stimulated with 0.7 mM H2O2 for 10 h (n = 4). B Cleavage of each caspase resulting from 0.7 or 1.1 mM H2O2 stimulation for 10 h, as detected by immunoblotting (n = 3). C Time course of caspase-9 cleavage resulting from 0.7 mM H2O2 stimulation, as detected by immunoblotting (n = 3). A *P < 0.05 by one-way ANOVA followed by Dunnett’s multiple comparisons test compared with WT cells with 0.7 mM H2O2 stimulation.

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: uPA knockdown affects ERK1/2 survival signaling. (a) Phosphorylation levels of ERK1/2, Akt and regulators of NF-κB signaling (IκBα) in transformed cells surviving TRAIL treatment. BJELR cells were either left untreated (‘control') or treated with TRAIL (1 μg/ml) during 7 or 16 h. Apoptotic cells (‘apop') were collected by successive washes and non-apoptotic attached cells (‘surv') were harvested for western blot analysis. Total and phosphorylated levels of ERK1/2 (Thr202/Tyr204), Akt (Ser 473) and IκBα (Ser32/36) were analyzed. Caspase-8 cleavage (‘Casp-8') was evaluated in each cell population. α-Tubulin, loading control. (b) Phosphorylation status of ERK1/2 and Akt upon uPA knockdown. BJELR cells were left non-transfected (‘mock') or transfected either with pooled siRNAs targeting PLAU mRNA (‘siPLAU') or non-targeting scramble siRNAs (‘scr'). Forty-eight hours after transfection, total and phosphorylated protein levels of ERK1/2 (Thr202/Tyr204) and Akt (Ser 473) were analyzed by western blot analysis. α–Tubulin, loading control. (c) Distribution of total (ERK1/2 Alexa488) and phosphorylated ERK1/2 (pERK1/2-Alexa 488; Thr202/Tyr204) in populations of BJELR cells untreated (‘mock'), scramble transfected (‘scr') and uPA depleted (‘siPLAU') was analyzed by flow cytometry. Images represent the distribution of fluorescence within each sample for a representative experiment out of three independent replicates. Isotypic IgG1 (‘IgG1') was used as control for background florescence. (d) Requirement of ERK1/2 signaling for cell survival upon TRAIL challenge. BJELR cells were either treated for 1 h with 20 μM U0126 MEK1/2 inhibitor or vehicle (DMSO). Apoptosis in untreated samples (‘U0126', ‘DMSO') and upon 3 h of treatment with 1 μg/ml of TRAIL (‘U0126+TRAIL', ‘DMSO+TRAIL') was analyzed by flow cytometry as the percentage of cells with positive labeling for cleaved PARP. Images correspond to one representative experiment out of four independent replicates. Percentage of cleaved PARP-positive cells is indicated in the upper right quadrant

Fig 5: DIABLO/SMAC mimetic loaded TNP induce autophagy-dependent apoptosis of HIV-1-infected macrophages. HIV-1-infected macrophages (HIV-Mφ) transduced with ATG5 siRNA (siATG5), ATG7 siRNA (siATG7), or scrambled siRNA (siNS) were exposed to vehicle, empty TNP (TNP) or DIABLO/SMAC mimetic loaded TNP (LCL-161 or AT-406 at 1 µM) for 4 h. Cells were then washed three times with PBS, and then incubated for a further 24 h in fresh media. (A) Left, representative western blots of ATG5 (ATG5-ATG12), ATG7, CASP8, cleaved CASP8 (cCASP), LC3B isoforms, PARP1, cleaved PARP1 (cPARP1), SQSTM1, and β-actin (ACTB). Right, densitometric analysis of blots. (B) Aliquots of supernatants were spectrophotometrically tested for LDH as a measure of cell death.