Nuclear Translocation of Procaspase-3 by TRB3 Figure 2. Cleavage of TRB3 promotes apoptosis along with CASP3 activation. (A and B) Twenty-four hours after transfection of HeLa (A) and Jurkat (B) cells with the indicated V5-TRB3 expression plasmid or control (ctrl) vector, the cells were treated with TNFa/CHX for 4 hr (HeLa cells) or anti-Fas antibody for 6 hr (Jurkat cells). The resulting dead cells were counted by trypan blue staining. Error bars indicate mean 6SD of three independent experiments. *P,0.05, **P,0.005, ***P,0.001, statistically significant difference. (C and D) Twenty-four hours after transfection with the indicated V5-TRB3 expression plasmid or control vector, HeLa (C) and Jurkat (D) cells were reseeded in 96-well plates (1.06104 cells/well), and then treated with TNFa/CHX and anti-Fas antibody, respectively, for the indicated times and CASP3/7 activity was then measured using the luminometric Caspase-GloH 3/7 Assay Kit. Each data point represents mean 6SD of three independent experiments. doi:10.1371/journal.pone.0042721.g002 ment and also by coexpression of WT-TRB3 (Figure 4D). The difference in the cytoplasmic/nuclear ratio of C163S-proCASP3- EmGFP between the tunicamycin-treated and WT-TRB3-over- expressed cells suggests that the nuclear translocation efficiency of C163S-proCASP3-EmGFP might be dependent on the TRB3 expression level. A similar phenotype was observed in D338A- TRB3 expressing cells (Figure S6). Using cells expressing HA-tagged inactive proCASP3 mutant (C163S-proCASP3-HA), we also obtained similar results of the localization before and after tunicamycin treatment (Figure S7). Therefore, we further examined whether the efficiency of nuclear translocation of C163S-proCASP3-HA would decrease by micro- RNA (miRNA)-mediated down-regulation of endogenous TRB3 expression. To this end, we designed two types of artificial miRNA targeting TRB3 mRNA (miR-TRB3-CDS and miR-TRB3- 39UTR), and which specifically suppressed endogenous TRB3 expression (Figure S8). We could identify the miRNA expressing cells by cocistronic expression of EmGFP from the same plasmid. The localization of C163S-proCASP3-HA was assessed in EmGFP expressing cells (Figure 4E and F). Consequently, the phenotype of cells positive for nuclear C163S-proCASP3-HA was decreased in miR-TRB3 expressing cells under tunicamycin- induced ER stress condition. Taken together, these results suggest that ER stress induced TRB3 expression leads to nuclear translocation of proCASP3. Nuclear proCASP3 did not Promote Apoptosis Results shown in Figure 3 and 4 suggested that TRB3 expression produced anti-apoptotic effect and it also induced nuclear translocation of proCASP3 under ER stress condition. We hypothesized that inhibition of apoptosis by TRB3 resulted from the nuclear translocation of proCASP3. Paradoxically speaking, the presence of proCASP3 in the cytoplasm, but not that in the nucleus, appears to be more important for the execution of apoptosis, because it has been proposed that the activation of CASP3 initially occurs in the cytoplasm, and the activated CASP3 then gets translocated to the nucleus [25]. To validate our hypothesis, we constructed three recombinant plasmids – one expressing EmGFP-tagged wild type proCASP3 (proCASP3- EmGFP), the second expressing nuclear localization signal (NLS) containing proCASP3-EmGFP (proCASP3-NLS-EmGFP) and the third expressing DsRed-NLS fusion protein (DsRed-NLS; control), and used them to investigate the effect of forced nuclear expression of proCASP3 on apoptosis. Accordingly, HeLa cells were separately transfected with these plasmids, and then the transfected cells were plated together in one glass bottom dish. After inducing apoptosis, EmGFP-fusion protein positive and DsRed positive cells were observed by live imaging. As expected, under normal culture condition (i.e. cells not fixed), proCASP3- EmGFP was localized in the cytoplasm (Figure 5A left panel), whereas proCASP3-NLS-EmGFP (middle panel) and DsRed-NLS (right panel) were localized only in the cell nucleus. The anti-Fas antibody was used for apoptosis induction, because it did not induce nuclear translocation of proCASP3 at least during imaging. As shown in Figure 5B and C, apoptotic cell death, based on cell morphology, was preferentially induced in cells expressing the cytoplasmic proCASP3 (proCASP3-EmGFP) than in cells expres- sing the nuclear proCASP3 (proCASP3-NLS-EmGFP) or the PLoS ONE | www.plosone.org 4 August 2012 | Volume 7 | Issue 8 | e42721