Nuclear Translocation of Procaspase-3 by TRB3 Figure 5. Nuclear proCASP3 did not promote apoptosis. (A–D) HeLa cells were separately transfected with the plasmid expressing EmGFP- tagged wild type proCASP3 (proCASP3-EmGFP), nuclear localization signal (NLS) containing proCASP3-EmGFP (proCASP3-NLS-EmGFP), or DsRed-NLS fusion protein (DsRed-NLS). After 24 hr, transfected cells were trypsinized and plated together in one glass bottom dish. After a further 24 hr, apoptosis was induced by the addition of anti-Fas antibody. Cells expressing the indicated fusion protein are shown (A). Behavior of the EmGFP- fusion protein positive and DsRed positive cells (same field at different times) was recorded by live imaging (B). Representative frames showing images of cells expressing the indicated fusion protein in a given field, as recorded in Figure 5B, are displayed (C). Scale bars = 20 mm (A and C), 50 mm (B). The number of apoptotic cells (based on cell morphology) was counted at 450 min after apoptosis induction by anti-Fas antibody (D). A minimum number of 30 cells were counted in a field for each type of fusion protein expressing cells. Four fields were randomly chosen for counting the number of apoptotic cells. Error bar: mean 6SD. *P,0.001. doi:10.1371/journal.pone.0042721.g005 then the mixture was incubated for 2 hr at 30uC. The assay using CASP9 was performed in the reaction mixture containing 10% PEG6000 because it is necessary for the CASP9 activity. Additionally, to remove PEG6000 from the reaction mixture after cleavage reaction, the biotinylated-TRB3 was recovered by streptavidin magnetic beads (Promega Corporation, Madison, WI, USA). These reaction mixture and recovered-TRB3 were boiled in SDS-PAGE sample buffer. The samples separated on SDS-PAGE were transferred to PVDF membrane (Millipore, Bedford, MA, USA). The membrane was probed with Alexa 488- conjugated streptavidin (Invitrogen), and then visualized using a Typhoon Imager (GE Healthcare, Piscataway, NJ). Cell Culture and Transfection HeLa and Jurkat cells were grown in Dulbecco’s modified eagle medium (DMEM) and RPMI medium, respectively. Each medium was supplemented with 10% fetal bovine serum (FBS), 100 units/ mL penicillin and 100 mg/mL streptomycin. Transient trans- fection of HeLa and Jurkat cells with plasmid was carried out using Lipofectamine 2000 (Invitrogen) and FuGENE6 (Roche, India- napolis, IN, USA), respectively, and following the manufacturer’s instructions. Empty vector pcDNA3.1 was used as a transfection control. Apoptosis Induction and Assay To induce apoptosis, cells were treated with TNFa/CHX [20 ng/mL TNFa (Calbiochem, La Jolla, CA), 100 mM CHX (Chemicon, Temecula, CA)], 125 ng/mL anti-Fas antibody (IgM, CH11) (Medical & Biological Laboratories Co., Ltd., Nagoya, Japan) or 5 mM tunicamycin (Sigma-Aldrich) for various times as indicated in the figure legends. For inhibition of apoptosis, the above reagents were supplemented with 100 mM z-VAD-FMK (Peptide Institute Inc., Osaka, Japan). DMSO was used as a treatment control. The treated cells were subjected to each assay or suspended in an equal volume of 0.5% trypan blue solution (Nacalai Tesque, Kyoto, Japan) for 1 min at room- temperature, and then the stained cells were counted as dead cells. Immunoblot Analysis After washing with PBS, cells were lysed in 26 SDS-PAGE sample buffer (125 mM Tris-HC1, pH 6.8, 20% glycerol, 4% SDS, 10% 2-mercaptoethanol, 0.001% bromophenol blue) and the proteins in the cell lysates were heat denatured. Proteins in the cell lysates were separated by SDS-PAGE and then transferred to PVDF membrane (Millipore) by electroblotting. Membranes were subsequently used in immunoblot assay using one of the following primary antibodies: anti-V5 epitope (#R960-25, Invitrogen), anti- TRB3 (#2488-1, Epitomics, Burlingame, CA), and anti-a-tubulin (#T9026, Sigma-Aldrich). Chemiluminescent signals generated by Immobilon Western HRP substrate Luminol Reagent (Millipore) or ImmunoStar (Wako, Osaka, Japan), were detected using an LAS-4000 mini biomolecular imager (GE Healthcare). Densitometric Image Analysis To measure the relative expression level of proteins in cells, acquired images were densitometrically analyzed by using the ImageJ software (NIH, Bethesda, MD, USA). Luminometric CASP3/7 Activity Assay CASP3/7 activity was measured by using the luminometric Caspase-GloH 3/7 Assay kit (Promega) and a GloMaxTM 96 Microplate Luminometer (Promega) according to the manufac- turer’s instructions. CASP3/7 activity shown was relative to the untreated control value. Knockdown of Endogenous TRB3 by Small Interfering RNA and microRNA Negative control siRNA (59-AATTCTCCGAACGTGT- CACGT-39) and siRNA oligonucleotide targeting 39UTR of human TRB3 (59-ATGAGGCTAGTTCTTGTCTAA-39) were purchased from QIAGEN (Valencia, CA, USA). Transfection of cells with siRNA was performed according to the manufacturer’s instructions using TransIT-siQUEST transfection reagent (Mirus Bio Corporation, Madison, WI). For knockdown of human TRB3 using a miRNA interference, DNA duplexes targeting the TRB3 coding sequence (CDS) (59-TTGGAGTTGGATGACAACTTA- 39) and 39UTR (59-CAGTTCCTGCTTGGGTGCTTA-39) were designed by using Invitrogen BLOCK-iT RNAi Designer (at www.invitrogen.com/rnaidesigner) and cloned into pcDNA6.2- GW/EmGFP-miR expression vector (Invitrogen) that enable identify artificial miRNA expressing cells by cocistronic expression of EmGFP. Negative control miRNA expression vector was purchased from Invitrogen. Immunofluorescence Analysis HeLa cells grown on coverslips were washed with cold PBS, and then fixed with 2% paraformaldehyde in PBS for 10 min. After washing with PBS, the fixed cells were permeabilized with 0.5% Triton X-100 (Nacalai Tesque) in PBS for 5 min. After washing with PBS, the cells were incubated in a blocking buffer [TBS containing 5% calf serum (Invitrogen)] for 1 hr. The cells were then incubated with one of the primary antibodies (listed below) in TBST containing 0.05% BSA for 1 hr at 37uC, washed three times with TBST (5 min each), and incubated with the Alexa Fluor 488/555-conjugated secondary antibody (Invitrogen) plus DAPI (Invitrogen) for 1 hr. After washing three times with TBST (5 min each), the stained cells were mounted on glass slides and visualized using a Carl Zeiss LSM710 confocal laser scanning microscope (Carl Zeiss, Jena, Germany). Primary antibodies: anti- V5 epitope (#R960-25, Invitrogen), anti-HA epitope (#11867423001, Roche), anti-Active-CASP3 (#9661, Cell Sig- naling Technology, Beverly, MA), and anti-TRB3 (#2488-1, Epitomics, Burlingame, CA). PLoS ONE | www.plosone.org 10 August 2012 | Volume 7 | Issue 8 | e42721