Nuclear Translocation of Procaspase-3 by TRB3 Figure 1. TRB3 is cleaved by caspases in vitro and in the apoptotic process. (A) Schematic representation of proteins expressed by the recombinant TRB3 constructs used in this study. WT, wild type TRB3; D338A, CASP3 cleavage-site mutant TRB3; and DC20, CASP3-cleaved form of TRB3. (B and C) Each recombinant TRB3 was synthesized and biotinylated using the wheat cell-free expression system as described in the Materials and Methods. The translation mixture was incubated with or without the indicated active caspase for 2 hr at 30uC. For the detection of N-terminal biotinylated-TRB3, the blot was probed with Alexa 488-conjugated streptavidin. DA denotes D338A-TRB3. (D) Twenty-four hours after transfection of HeLa and Jurkat cells with the indicated V5-tagged TRB3 expression plasmid, cells were treated with TNFa (20 ng/mL)/CHX (100 mM) (HeLa cells) or anti-Fas antibody (125 ng/mL) (Jurkat cells) in the absence or presence of z-VAD-FMK (100 mM) for 4 hr. DMSO was used as a treatment control. The cell lysates were subjected to immunoblot analysis using anti-V5 antibody to detect the N-terminal V5-tagged TRB3. a-Tubulin was used as an internal control. doi:10.1371/journal.pone.0042721.g001 expression in cells under ER stress condition (Figure 3F). As shown, TRB3 siRNA (siTRB3) specifically reduced the expression of endogenous TRB3 upto 24 hours following the tunicamycin treatment (Figure 3G), by which time the relative activity of CASP3/7 increased over the activity found in negative control siRNA (siNC)-transfected cells (Figure 3H). Taken together, these results strongly suggest that TRB3 intrinsically blocks CASP3 and/or CASP7 activation and subsequent apoptosis under TRB3 non-cleavable conditions, such as ER stress. TRB3 Expression Induces Nuclear Translocation of proCASP3 In Figure 3, we have presented evidence that suggests that TRB3 negatively regulates the activation of CASP3. However, the premature CASP3 (i.e. procaspase-3; proCASP3) and TRB3 are differently localized in cells; proCASP3 is predominantly localized in the cytoplasm [23,24], whereas TRB3 is localized in the nucleus [5,7]. Thus, even though TRB3 is a substrate for CASP3, how these two molecules would interact with each other still remains elusive. To gain further insights about that, we next examined whether the subcellular localization of proCASP3 would be affected by TRB3 expression. For this purpose, we constructed a recombinant plasmid expressing an EmGFP-tagged inactive proCASP3 mutant (C163S-proCASP3-EmGFP), in which the active site cysteine residue was altered to a serine residue (C163S) to prevent apoptosis. We next cotransfected HeLa cells with the C163S-proCASP3-EmGFP and WT-TRB3 expression plasmids, and subsequently assessed the subcellular localization based on the relative fluorescence intensity of EmGFP by using confocal microscopy. Under normal culture condition, C163S-proCASP3- EmGFP predominantly exhibited cytoplasmic localization (left panels in Figure 4A and B, see arrowheads), which is consistent with the known localization of proCASP3. Interestingly, C163S- proCASP3-EmGFP was found mainly in the nucleus of cells coexpressing WT-TRB3 (Figure 4A, see arrow). As shown in Figure 3A, under normal culture condition cells maintained the endogenous TRB3 expression at a relatively low level, which significantly increased after tunicamycin treatment for 8 hours. So next, we examined the localization of C163S- proCASP3-EmGFP under tunicamycin-induced ER stress condi- tion. Because active CASP3 was hardly detected by immunoflu- orescence in tunicamycin-treated morphologically normal cells (98.7% were active CASP3 negative; 100 cells were assessed in three independent experiments), we assumed that C163S-pro- CASP3-EmGFP as the premature form (i.e. proCASP3) in these cells (Figure S5). Increased amount of C163S-proCASP3-EmGFP was found in the nucleus of tunicamycin-treated cells (Figure 4B right panel, see arrowheads). Furthermore, we could monitor the nuclear accumulation of C163S-proCASP3-EmGFP by live imaging of cells up to 24 hours following the tunicamycin treatment (Figure 4C). The number of cells expressing nuclear C163S-proCASP3-EmGFP was increased by tunicamycin treat- PLoS ONE | www.plosone.org 3 August 2012 | Volume 7 | Issue 8 | e42721