Fig 2: Effect of different caspase inhibitors on STAT1 activation and STAT1 target gene expression in CD95 stimulated cells. (A,B) Western blot analysis of MCF-7 cells treated with either DMSO solvent control, 20 μM of an inhibitor of caspase-2 (zVDVAD), caspase-3/7 (zDEVD), caspase-6 (zVEID), caspase-8 (zIETD), caspase-9 (zLEHD), or caspase-10 (zAEVD) upon LzCD95L (A) or anti-APO-1 (B) treatment for 4 days. All uncropped immunoblot images are included in Fig. S9. (C) Real-time PCR quantification of mRNAs in MCF-7 cells treated as shown in A upon exposure to LzCD95L for 4 day. (D) Real-time PCR quantification of mRNAs in MCF-7 cells treated as in A. Error bars represent the SD of three biological replicates. (E) Real-time PCR quantification of mRNAs in MCF-7 cells treated as in A. Student’s t-test was performed compared to matching control. A linear model for continuous gene expression levels, using binary predictors for LzCD95L and zVDVAD or zIETD and their interaction term, was used to evaluate whether the effect of LzCD95L on gene expression varied depending on the presence of zVDVAD or zIETD (red asterisks). p-value *<0.05, **<0.001; ***<0.0001; ns, not significant.

Fig 3: Apoptotic mechanism. (a,b) Percentage of apoptosis and representative dot plots of annexin V-FITC and PI-stained cells analyzed by flow cytometry. Data are expressed as mean ± SD of n = 4 experiments. At least 10.000 events were acquired. (c–h) Protein expression levels of caspase 3, caspase 9, PARP, Bax and Bcl-2 from LoVo cells treated for 72 h with milk (40% v/v), δVB (2 mM), milk+δVB, or HBSS-10 mM Hepes (40% v/v) (Ctr). Lane 1 = Ctr, lane 2 = milk, lane 3 = δVB, lane 4 = milk + δVB. Analysis of densitometric intensity was calculated with Image J 1.52n version software. Arbitrary units of protein expression (AU) were quantified using α-tubulin or β-actin. Antibodies against Bax, Bcl-2 and SIRT6 (reported in Fig. 4) were blotted on the same filter and quantified by using the same loading control (α-tubulin). (i,j) Flow cytometric analysis and representative dot plots of annexin V-FITC and PI double staining LoVo cells treated with caspase 9 inhibitor Z-LEHD-FMK (40 μM) or chloroquine (50 μM). Data are expressed as mean ± SD of n = 3 experiments. At least 10.000 events were acquired. (k) Cleaved caspase 3 protein expression level from LoVo cells treated for 72 h with milk+δVB, Z-LEHD-FMK + milk+δVB or HBSS-10 mM Hepes (40% v/v) (Ctr). Lane 1 = Ctr, lane 2 = milk + δVB, lane 3 = Z-LEHD-FMK + milk+δVB. (l) Protein expression levels of caspase 8 in LoVo cells after 72 h of treatment with milk (40% v/v), δVB (2 mM), milk+δVB, or HBSS-10 mM Hepes (40% v/v) (Ctr). Lane 1=Ctr, lane 2 = milk, lane 3 = δVB, lane 4 = milk + δVB. *P < 0.05 vs Ctr, **P < 0.01 vs Ctr, †P < 0.05 vs milk, ††P < 0.01 vs milk, +P < 0.05 vs milk+δVB, ++P < 0.01 vs milk+δVB. The full-length blots are included in the supplementary information (Fig. S4).

Fig 4: VSV-S Induces Apoptosis by Intrinsic Apoptotic Pathway(A) Cell viability of MDA-MB-231 cells 36 h after infection by VSV-S versus wtVSV at different MOIs. (B) Immunoblot analysis of cleaved PARP, cleaved caspase-9, and cleaved caspase-3 in MDA-MB-231 cells. VSV-S infection induced apoptosis in MDA-MB-231 cells. wtVSV infection was used for the comparison, and uninfected cells were used as a control. (C) Cell viability of MDA-MB-231 cells 36 h after infection by VSV-S, in the presence of an inhibitor of caspase-8, caspase-9, or caspase-3. Error bars represent mean ± SEM. *p < 0.05, ***p < 0.001 by one-way ANOVA followed by post hoc Tukey’s for multiple comparisons.

Fig 5: MEK1 is cleaved in a caspase‐dependent manner during apoptosis. (A) U2OS and A375 cells were treated with etoposide (60 μm for 24 h) and the expression level of endogenous MEK1 was analyzed by immunoblotting using an antibody against the N‐terminal region (amino‐acid residues 2–18) of MEK1. Etoposide induced the appearance of the cleaved 35‐kDa form of MEK1 (Cleaved). PARP, BRaf, MEK2, and ERK1/2 in cell extracts were also probed with the appropriate antibodies. Actin served as a loading control. Short, short exposure. Long, long exposure. FL, full‐length. *, nonspecific bands. (B) U2OS cells were stimulated with etoposide (60 μm) for the indicated times. The cleavage of MEK1 and PARP was analyzed by immunoblotting. (C, D) A549 (C) and Jurkat (D) cells were exposed to osmotic stress (0.6 m sorbitol) or anti‐Fas antibody (200 ng·mL−1) for the indicated times. Immunoblotting was performed as in (A). (E, F) U2OS (E) and Jurkat (F) cells were stimulated with etoposide (60 μm for 24 h), osmotic stress (0.6 m sorbitol for 6 h), or anti‐Fas antibody (200 ng·mL−1 for 8 h) as indicated. The induced proteolytic cleavage of PARP and MEK1 was analyzed by immunoblotting. (G) U2OS and Jurkat cells were stimulated with etoposide (60 μm for 24 h) or anti‐Fas antibody (200 ng·mL−1 for 10 h) as indicated. The cleavage of MEK1, caspase‐9, caspase‐8, and caspase‐3 was analyzed by immunoblotting using appropriate antibodies. In (A, E–G), cells were pretreated with (+) or without (−) a pan‐caspase inhibitor Z‐VAD‐FMK (100 μm), a caspase‐8 inhibitor Z‐IETD‐FMK (20 μm), or a caspase‐9 inhibitor Z‐LEHD‐FMK (20 μm) for 2 h before stimulation as indicated.