Quantification of C163S-proCASP3-EmGFP and C163S- proCASP3-HA Localization Preferential localization of C163S-proCASP3-EmGFP in the fixed cells was assessed as either mainly cytoplasmic (C) or mainly nuclear (N) based on their observed relative fluorescence intensity using a Carl Zeiss LSM710 confocal laser scanning microscope. In a similar way, the localization of C163S-proCASP3-HA was also assessed in artificial miRNA expressing cells that could be assumed by cocistronic expression of EmGFP. To rule out the contribution of the active CASP3 to the quantification of proCASP3 localization, we examined only morphologically normal cells (98.7% of tunicamycin-treated morphologically normal cells were active CASP3 negative; see text and Figure S5). Live Imaging Analysis Transfected HeLa cells grown on a glass bottom dish were set in the culture environment (37uC, 5% CO2) of microscope after treatment with a given agent. Images of EmGFP-fusion protein positive and DsRed positive cells, respectively, were acquired using the Carl Zeiss LSM710 confocal laser scanning microscope equipped with LSM 7 live module. Time-lapse images were taken with a 636plan apochromatic objective with a numerical aperture of 1.4. Each frame of time-lapse images was acquired every 5 minutes (Movie S1) or 10 minutes (Figure 4C) for the indicated times. Movie S1 was shown at 10 frames/sec. Statistics Data shown are mean 6S.D. Student’s t test was used to determine the significance of differences. P values ,0.05 were considered to be statistically significant. Supporting Information Figure S1 Sequence alignment analysis of TRB3 homo- logs. Organisms, more than 50% amino acid identity to human TRB3, were selected in this analysis. The result showed that TRB3 caspase-cleavage site is highly conserved. Although, homology of Danio rerio TRB3 is relatively low (52.9%), it should be noted that the P4-P1 substrate recognition motif (VVPD) is completely conserved. This raises a possibility that TRB3 is cleaved in various species and the cleavage has biological significance. Sequence alignment was carried out using the Geneious software. Organism, Accession number: Homo sapiens, AAH27484; Pongo abelii, XP_002830205; Macaca mulatta, XP_002798226; Callithrix jacchus, XP_002747457; Canis lupus familiaris, XP_542943; Ailuropoda melanoleuca, XP_002925440; Bos taurus, NP_001069571; Oryctolagus cuniculus, XP_002710903; Mus musculus, NP_780302; Rattus norvegicus, NP_653356; Danio rerio, NP_998034 (PDF) Figure S2 TNFa/CHX- and anti-Fas antibody-induced cell death was strongly inhibited by the caspase in- hibitor z-VAD-FMK. (A, B) Twenty-four hours after trans- fection with the V5-WT-TRB3 expression plasmid, HeLa (A) and Jurkat (B) cells were treated with TNFa (20 ng/mL)/CHX (100 mM) for 4 hr and anti-Fas antibody (125 ng/mL) for 6 hr, respectively, in the absence or presence of z-VAD-FMK (100 mM). The resulting dead cells were counted by trypan blue staining. Error bars indicate mean 6SD of three independent experiments. (PDF) Figure S3 Cleavage of endogenous TRB3 in apoptotic cells. HeLa cells were treated with tunicamycin (5 mM) for 8 hr, and then treated with TNFa/CHX in the absence or presence of Nuclear Translocation of Procaspase-3 by TRB3 z-VAD-FMK (100 mM) for 3 hr. DMSO was used as a treatment control. The cell lysates were subjected to immunoblot analysis using anti-TRB3 antibody. Cleaved PARP (#9541, Cell Signaling Technology) is a marker of apoptosis. a-Tubulin was used as an internal control. (PDF) Figure S4 Tunicamycin-induced cell death was strongly inhibited by the caspase inhibitor z-VAD-FMK. Twenty- four hours after transfection with the control vector, HeLa cells were treated with tunicamycin for 36 hr in the absence or presence of z-VAD-FMK. The resulting dead cells were counted by trypan blue staining. Error bars indicate mean 6SD of three independent experiments. (PDF) Figure S5 Active CASP3 was hardly detected in tunica- mycin-treated morphologically normal cells. HeLa cells grown on coverslips were treated with tunicamycin for 8 hr. The fixed cells were stained with anti-Active-CASP3 antibody (red), and counterstained with DAPI (blue) and Alexa 488-conjugated phalloidin (green) to visualize the nuclei and cell morphology, respectively. 100 cells were assessed in three independent experi- ments, and 98.7% of tunicamycin-treated morphologically normal cells were active CASP3 negative. Scale bars = 20 mm. (PDF) Figure S6 Nuclear translocation efficiency of proCASP3 mediated by D338A-TRB3 was almost same as that by WT-TRB3. (A) HeLa cells grown on coverslips were cotrans- fected with the EmGFP-tagged inactive proCASP3 mutant (C163S-proCASP3-EmGFP) and V5-D338A-TRB3 expression plasmids, and then incubated for 24 hr. The fixed cells were stained with anti-V5 antibody (red) and counterstained with DAPI (blue) to visualize the nuclei. Scale bars = 20 mm. (B) Twenty-four hours after transfection, HeLa cells grown on coverslips were fixed, and then stained as described above. Localization of C163S- proCASP3-EmGFP in cells that are also expressing TRB3 from the respective plasmid was quantified as cytoplasmic, mainly cytoplasmic or cytoplasmic equal to nuclear (C, C.N, C = N), or as nuclear or mainly nuclear (N, N.C). Over 30 cells were assessed in three independent experiments. Error bar: mean 6SD. (PDF) Figure S7 The localization of C163S-proCASP3-HA. HeLa cells grown on coverslips were transfected with the C163S-proCASP3-HA expression plasmid. After 24 hr, the cells were incubated with or without tunicamycin for 8 hr. The localization of C163S-proCASP3-HA was observed by immuno- fluorescence staining with an anti-HA antibody (red). Inner panel was merged with DAPI (blue) to visualize the nuclei. Scale bars = 20 mm. (PDF) Figure S8 Artificial miRNAs targeting TRB3 mRNA specifically suppress endogenous TRB3 expression. HeLa cells grown on coverslips were transfected with the indicated artificial miRNA expression plasmid that enable identify the miRNA expressing cells by cocistronic expression of EmGFP. After 24 hr, the cells were treated with tunicamycin for 8 hr, and then fixed. Endogenous TRB3 and nuclei were visualized by immunofluorescence staining with an anti-TRB3 antibody (red) and DAPI (blue) respectively. Scale bars = 20 mm. miR-NC denotes negative control miRNA. (PDF) PLoS ONE | www.plosone.org 11 August 2012 | Volume 7 | Issue 8 | e42721