HeLa and A549 cells were maintained in Dulbecco's modified Eagle's medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum and antibiotics (final concentration, 10,000 U/ml penicillin and 10 mg/ml streptomycin; Wako Pure Chemical Industries, Ltd., Osaka, Japan). The cells were allowed to adhere and proliferate for ≥24 h at 37°C in 5% CO₂ prior to the following experiments.

The preparation of nucleoplasmic and cytoplasmic fractions was performed as previously described (21). NE-PER nuclear and cytoplasmic extraction reagent (Pierce, Thermo Fisher Scientific, Inc., Rockford, IL, USA) was used according to the manufacturer's protocol, and prepared fractions were denatured with 2X Laemmli Sample buffer (Bio-Rad Laboratories, Inc., Hercules, CA, USA) for western blot analysis.

The procedures for whole lysate preparation and western blot analysis have been described (22). Protein concentration of the lysates was measured by the Bradford method. In brief, denatured samples (25 µg) were applied to 10% acrylamide gels. Following SDS-PAGE, gels were transferred to polyvinylidene difluoride membranes and blocked with 5% skim milk for 1 h. Then, blocked membranes were subjected to antibody binding. The first antibodies were rabbit anti-human Upf2 polypeptide antiserum (prepared in our laboratory), monoclonal mouse anti-human lamin A/C antibody (1:1,000; cat. no. sc-7292; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and monoclonal rabbit anti-human caspase 3 (1:1,000; cat. no. 9665; Cell Signaling Technology, Inc., Danvers, MA, USA). In addition, monoclonal mouse anti-RBM8A antibody was purchased from Sigma-Aldrich (1:500; 4C4 clone; cat. no. Y1253). β-actin (ACTB) was used as the reference protein, and was detected using mouse monoclonal anti β-actin antibody purchased from Sigma-Aldrich (1:5,000; AC 15 clone; cat. no. A5441). Incubation with primary antibody were performed at 4°C, overnight. Primary antibodies were detected with horseradish peroxidase-conjugated polyclonal goat anti-mouse (1:5,000; cat. no. P0447) or anti-rabbit (1:5,000; cat. no. P0448) secondary antibodies (Dako, Glostrup, Denmark).

One day prior to the small interfering RNA (siRNA) transfection experiments, HeLa cells were seeded on to culture plates or dishes. The depletion of Upf2 was performed using stealth RNA interference (RNAi) molecules (HSS178005), and the knockdown of RBM8A was achieved using HSS115052; the two siRNAs were obtained from Invitrogen™; Thermo Fisher Scientific, Waltham, MA, USA). Two double-stranded molecules of the Stealth RNAi^® negative control kit (Thermo Fisher Scientific) and medium GC duplex were used as the negative control. Transfections were performed using Lipofectamine™ RNAiMAX transfection reagent (Thermo Fisher Scientific), according to the manufacturer's protocol.

The full details of the procedure followed for immunostaining were previously described (23). Briefly, HeLa cells were washed three times with phosphate-buffered saline (PBS) and fixed at room temperature with 4% paraformaldehyde solution (Taab Laboratory Equipment Ltd, Aldermaston, Reading, UK) in PBS for 10 min. After washing three times with PBS, the cells were treated with 0.2% Triton X-100 (Sigma-Aldrich)/PBS for 10 min at room temperature. Samples were washed three times with PBS. For the ribonuclease (RNase) treatment experiments, cells were fixed with cold ethanol for 10 min and treated with 10 ng/ml RNaseA solution (Qiagen GmbH, Hilden, Germany) at 37°C for 30 min, followed by three further washes with PBS. Blocking was performed with 1% bovine serum albumin (BSA; Wako Pure Chemical Industries, Ltd.) with gentle agitation for 30 min at room temperature. Following this procedure, the cells were incubated with the first antibodies, rabbit anti-human Upf2 polypeptide antiserum prepared in our laboratory), anti-human lamin A/C antibody (Santa Cruz Biotechnology, Inc.), mouse anti-RBM8A antibody purchased from Sigma-Aldrich (4C4 clone) or rabbit anti-RBM8A antibody (prepared in our laboratory) with gentle agitation for 2 h at room temperature. Binding of the first antibody was detected using Alexa Fluor 488- or 594-conjugated secondary antibodies (Molecular Probes Life Technologies, Carlsbad, CA, USA) with gentle agitation at room temperature for 60 min, and nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). After washing the cells with PBS three times, ProLong^® Gold Antifade reagent (Invitrogen™; Thermo Fisher Scientific) was used to prevent fading. Images were captured with either an Axiovert 200 M inverted fluorescence microscope or an LSM 710 confocal point-scanning microscope (Carl Zeiss, München, Germany). ZEN 2008 SP1.1 software (Carl Zeiss) was used to analyze signal intensity.

To confirm the association between mRNA and Upf2, HeLa cells were collected using a cell scraper and suspended in cold NET-2 buffer [50 mM Tris-HCl (pH 7.4), 300 mM NaCl, 0.05% Igepal-CA630; Sigma-Aldrich]. For nuclear fractions, cells were treated with cNE-PER nuclear and cytoplasmic extraction reagent (Thermo Fisher Scientific, Inc.) and nuclear fractions were collected. Subsequently, nuclear fractions were suspended in cold NET-2 buffer. The samples were sonicated using the Vibra cell sonicator (Sonics & Materials, Inc., Newtown, CT, USA), and the sonicated samples were centrifuged for 15 min at 15,000 × g. The supernatant was treated with anti-Upf2 antiserum and normal rabbit serum as the control. Following an incubation with agitation in a cold room, Protein G-conjugated agarose beads (Invitrogen™; Thermo Fisher Scientific) were added to the samples, and the samples thus obtained were again incubated in a cold room. Following sufficient washing with NET-2 buffer, the beads were resuspended in Laemmli sample buffer (Bio-Rad Laboratories, Inc.) with 2-mercaptoethanol (Sigma-Aldrich). The sample was boiled, and the resultant sample buffer solution was processed for western blotting, which was performed as described above.

The direct observation of Upf2-EJC complex formation was performed using a Duolink^® kit (Olink Bioscience, Uppsala, Sweden; product now owned by Sigma-Aldrich) following the manufacturer's protocol (23). This method enables the visualization of complex formation in cells by proximity ligation of single-stranded DNA conjugated with a secondary antibody (24), and is available for the complex in nuclei and cytoplasm (25). Signal can be observed when the distance between the secondary antibodies is <40 nm. Therefore, this method can detect not only direct protein-protein interactions, but also complex formation, assuming that the bound first antibodies are proximal enough. The first antibodies used in the present study were as described in the above Immunostaining and observation section. Images were captured with either an Axiovert 200 M inverted fluorescence microscope or an LSM 710 confocal point-scanning microscope (Carl Zeiss, München, Germany).

In order to assess whether Upf2 was resident in the nuclei, HeLa cells were fractionated into their respective nuclear and cytoplasmic fractions. These fractions were analyzed using western blotting. The purity of each fraction was evaluated by blotting with anti-lamin A/C (nuclear marker) and anti-caspase 3 (cytoplasmic marker) antibodies. Almost all the lamin A/C was detected in the nuclear fraction, and caspase 3 was predominantly detected in the cytoplasm. Therefore, it was confirmed that the fractionation process had been successful, and Upf2 was clearly detected in the two fractions (Fig. 1).

Subsequently, to confirm the results of western blotting, immunostaining experiments were performed. Previous reports have revealed the cytoplasmic and perinuclear localization of endogenous or exogenous Upf2 (11,13). In accordance with these reports, the results in the present study also demonstrated that Upf2 is predominantly localized to the cytoplasmic perinuclear region, although a limited quantity of signal was observed in the nucleus (Fig. 2A). Subsequently, knockdown experiments were performed to confirm the specificity of the obtained signals. The Upf2-specific siRNA, prepared in our own laboratory, successfully depleted the Upf2 signals in the perinuclear and intranuclear regions (Fig. 2A). The knockdown efficiency was confirmed by western blotting of the siRNA-treated and control siRNA-treated cells (Fig. 2B). These findings suggested that the signals from immunostaining observed in the present study had originated from the Upf2 molecules. Thus, it was deduced that Upf2 proteins exist not only in the perinuclear region, but also in the nucleoplasmic region.

To analyze which factors bind to the RNA molecule, RNase treatments are available and have been used in a number of previous studies (26–28). To examine whether Upf2 protein binds to RNA molecules in cells, methanol-fixed cells were treated with RNaseA solution. Following washing of the cells, cells were stained with anti-lamin A/C and anti-Upf2 antibodies, as described in the Materials and methods section. In addition, RBM8A was also stained as a control for the RNase treatment. As shown in Fig. 3, the majority of Upf2 in the cytoplasm and the nucleoplasm disappeared on RNase treatment. This suggested that appreciable quantities of Upf2 bind to the mRNA molecules in the nucleoplasm, as found for the cytoplasm and the perinuclear region. The signal from lamin A/C remained, and this clearly demonstrated that the structural integrity of the nucleus was not affected by the RNase treatment.

The presence of Upf2-binding RBM8A in the nuclear fraction was subsequently investigated. Following fractionation, Upf2 protein was immunopurified using a specific antibody. RBM8A was detected to a limited extent in the immunoprecipitate of the nuclear fraction, similarly to the cytoplasmic fraction (Fig. 4).

Finally, to verify the interaction between Upf2 and EJC in the nucleoplasm, a proximity ligation in situ assay with rabbit anti-Upf2 and mouse anti-RBM8A antibodies (a component of EJC) was performed (7–10,29). Signals from the proximity ligation in situ assay were detected under a fluorescence microscope from the nuclei and the cytoplasm. In addition, knockdown of either the Upf2 or the RBM8A gene resulted in a reduction in signal intensity (Fig. 5). Under a confocal laser scanning microscope, sliced images were obtained, and the images revealed the nuclear-localized signals in addition to cytoplasmic signals (data not shown). These findings were not cell-type-specific, since similar results were obtained with human A549 cells under identical conditions. These results suggested that the Upf2 protein resides proximally to RBM8A in the nuclei and cytoplasm, and is included in the EJC.