Fig 2: Baseline computational model of Yoda1 and TRAIL synergy.a Schematic of calcium and TRAIL-mediated apoptosis. Dark red coloring indicates additions to the computational model. b Apoptosis or cPARP concentration of cancer cells treated with TRAIL with or without Yoda1. Dashed line represents the threshold of cPARP at which cancer cells are considered apoptotic. c Time of MOMP determined by release of Smac, which follows MOMP for cancer cells treated with TRAIL with or without Yoda1. d Apoptosis of cancer cells treated with Yoda1 with or without TRAIL. e Time of MOMP of cancer cells treated with Yoda1 with or without TRAIL

Fig 3: M. tb infection does not modify pro-apoptotic proteins. Human MDMs were obtained after seven days in culture. 2 × 106 MDMs were infected at MOI 1 and MOI 10 with an avirulent (H37Ra) and MOI 1 and MOI 5 with a virulent (H37Rv) strain of M. tb; 2 × 106 MDMs were not infected as a control (Uninf). At 24 h postinfection, cells were recovered and prepared for Western Blot. Representative Western blot of BAX, DIABLO, BAK, and GAPDH (a). Band densities of BAX (b), DIABLO (c), and BAK (d) were normalized against and quantified by densitometry analysis with the ImageJ software. Results are shown in relative units (RU) of concentration. The bar graphs show the mean ± SD from two independent experiments (n = 2 donors and two technical replicates). Statistical analysis was performed using Kruskal–Wallis analysis, followed by Dunn’s post hoc test.

Fig 4: ZFAS1 regulates APL cell apoptosis through the modulation of Bcl-2 family proteins. (A) APL cells were transfected with si-NC, si-ZFAS1-1 or si-ZFAS1-2 for 48 h. The levels of Bcl-2, Mcl-1, XIAP and Bax were examined by western blot analyses with the indicated antibodies. Densitometry analysis of the western blots is presented on the right. **P<0.01, ***P<0.001 vs. si-NC. (B) APL cells were transfected with si-NC, si-ZFAS1-1 or si-ZFAS1-2 for 48 h. Cytosolic fractions were subjected to western blotting with the indicated antibodies and densitometry analysis is presented on the right. ***P<0.001 vs. si-NC. Values are presented as the mean ± standard deviation (n=3). ZFAS1, long non-coding RNA zinc finger antisense 1; APL, acute promyelocytic leukemia; si- small interfering RNA; NC, negative control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Bcl-2, B-cell lymphoma-2; Mcl-1, induced myeloid leukemia cell differentiation protein Mcl-1; XIAP, E3 ubiquitin-protein ligase XIAP; Bax, apoptosis regulator BAX; Smac/DIABLO, Diablo homolog mitochondrial.

Fig 5: tBID induces MOMP and apoptosis in HCT AKO cells Quantification of the effect of individual BCL‐2‐family proteins on HCT AKO cell death, measured as percentage of cells showing released cyt c or pyknotic nuclei. Data correspond to at least three independent experiments, n > 30 cells per condition per experiment. ***P < 0.001 and *P < 0.05 with respect to GFP condition.Representative confocal immunofluorescence and Brightfield (BF) images of an apoptotic HCT AKO cell expressing tBID‐GFP (green), showing cyt c release (magenta), formation of pyknotic nuclei (cyan) and plasma membrane blebs. Scale bar is 10 µm.Western blot analysis of SMAC and cyt c release in GFP‐BAX, tBID‐GFP and GFP‐BCLXL expressing cells at 12, 16 and 24 h after transfection in HCT AKO. SN (supernatant, cytosolic fraction), P (pellet, membrane fraction) and PS (Ponceau Staining).Quantification of caspase activity in HCT AKO cells expressing GFP‐BAX, GFP‐BAK, tBID‐GFP, GFP‐BCLXL and GFP at different time points after transfection. n = 3 independent experiments. ***P < 0.001, **P < 0.025 and *P < 0.05 with respect to GFP condition. Error bars represent SD.Representative western blot analysis of PARP1 cleavage upon tBID‐GFP expression in HCT AKO cells. n = 3 independent experiments.Kinetics of the expression of tBID‐GFP (blue) and PI intake upon tBID expression (purple) and untransfected HCT116 AKO cells (in grey). Plots show the average fluorescence intensity (dark lines) and its standard deviation (shaded areas) from three technical replicates and three independent experiments. Fluorescence intensity was obtained by analysing the total fluorescence per well using incucyte system every 60 min.Quantification of PI+ cells from total transfected cells with GFP‐BAX, tBID‐GFP, GFP‐BCLXL and GFP. n = 3 independent experiments with > 10,000 cells per condition per experiment. ***P < 0.001 with respect to untransfected condition.Cell death induced by tBID‐GFP in HCT AKO cells in the absence and presence of ZVAD, NSA and Ferrostatin‐1. Data correspond to three independent experiments, n > 30 cells per condition per experiment. ***P < 0.001 with respect to MOCK condition. Data information: In (A), (G) and (H), dots correspond to independent experiments, boxes represent 96% confidence interval, the average is represented by the line inside the box and whiskers correspond to SD. The statistical significance was assessed by one‐way analysis of variance (ANOVA). Source data are available online for this figure.