Fig 2: Sub-lethal oxidative stress induces the activation of caspase 3L6 cells were exposed to 50μM H2O2 or 1μM STS for the indicated time points before A. Western Blot analysis of caspase 3 cleavage and B. Caspase 3 activity was assessed, statistical analysis was done by comparing treatment (H2O2 or STS) to untreated control at respective time point (24h or 48h). Values represent mean +/- SEM, *P < 0.05; n = 4 (t-test). C. Detection of cleaved caspase 3 by immunofluorescence analysis of cells exposed to 50μM H2O2 for 24h, as viewed under a confocal microscope with low magnification (scale = 50μm) and high magnification (scale = 30μm). D. Analysis of the nuclear localization of cleaved caspase 3 shown in (C). Values represent mean +/- SEM, ***P < 0.0005; control: n = 49, treated: n = 73 (t-test). E.-G. Western Blot analysis of the cleavage of classical caspase 3 substrate in cells exposed to 50μM H2O2 or 1μM STS: (E) Gelsolin, (F) Lamin A/C and (G) PARP.

Fig 3: Activation of caspase 3 and lysosome biogenesis induced by subtoxic oxidative stress involves ironL6 cells were incubated with the iron chelators DFO (50μM) and DFP (50μM), before being exposed to 50μM H2O2. A. Values represent mean +/- SEM, *P < 0.05; n = 9 (t-test). The inhibitory effect of DFO and DFP on LMP was statistically significant, P < 0.05 (mixed model). B. Values represent mean +/- SEM, *P < 0.05; n = 5 (t-test). The inhibitory effect of DFO and DFP on caspase 3 activity was statistically significant, P < 0.05 (mixed model). C. TFEB cellular location in presence or absence of DFO and DFP (scale = 30μm), D. mRNA expression of TFEB target genes, Lamp2, Ctsb, Ctsl, Neu1, Atg9b and Sqstm1, and E. lysosome number in presence or absence of DFO and DFP was assessed as described in materials and methods. Values represent mean+/- SEM, *P < 0.05, **P < 0.005; n = at least 3 independent experiments (t-test). The inhibitory effect of DFO and DFP was statistically significant for P < 0.05 (mixed model). F. Caspase 3 activity in cells exposed to 1μM STS, pre-treated with DFO and DFP. Values represent mean+/- SEM, *P < 0.05; n = 5, (t-test). The effect of DFO and DFP was not statistically significant, P>0.05 (mixed model).

Fig 4: The proteolytic cleavage of Sp3 in the viral lytic cycle is dependent on caspase activation. PEL cells lines including Tet-On-F-ORF50 (A), HH-B2 (B), and BCBL-1 (C) were preincubated with or without a pan-caspase inhibitor Z-VAD-FMK (100 μM) for 1 h, followed by cotreatment with doxycycline, SB, and SB plus TPA, respectively. At the indicated time points (24, 48, and 72 h) after treatment, cells were prepared and analyzed for the expression of cleaved PARP, cleaved caspase-3 (CC3), and Sp3 by Western blotting. The untreated cell samples (lane 1) and cell samples receiving only Z-VAD-FMK (lane 5), which were harvested after 24 h of culture, served as control groups.

Fig 5: 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.