The U937T cells provided by Dr Tenen from the Harvard Institute of Medicine (Harvard Medical School, Boston, MA, USA) (22) were U937 cells stably transfected with a pUHD-tetracycline-responsive transcription activator (tTA) under the control of a tetracycline-inducible promoter. The U937T cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 1 mg/ml tetracycline and 0.5 mg/ml puromycin (Sigma-Aldrich, St. Louis, MO, USA).

Full-length (wild type and D163A mutant) and fragment (1-163) of ANP32B cDNA with flag tag, a polypeptide protein tag with sequence motif DYKXXD, were amplified from the plasmid which was generated as previously described (12), and then cloned and inserted into the pTRE2hyg expression vector (BD Clontech, Palo Alto, CA, USA), a tetracycline-responsive expression vector, in order to form the corresponding plasmids. The sequence of the cDNA insert of the plasmid was confirmed by sequencing (Applied Biosystems 3730/3730xl DNA analyzer, Sangon Biotechnology, Shanghai, China). Following which, the plasmids were transfected into the U937T cells at passage 5 (provided by Dr DG Tenen at Harvard Institutes of Medicine, Harvard Medical School, Boston, MA, USA) containing stably transfected pUHD-tTA (BD Clontech, Palo Alto, CA, USA), whose expression is turned off in the presence of tetracycline (Calbiochem, San Diego, CA). In principle, the expression of tetracycline-responsive transcription activator (tTA) and target proteins should be extremely low in tetracycline-containing medium. In the absence of tetracycline, tTA activates its own promoter to produce more tTA which in turn induces the expression of target proteins (23). To generate the corresponding stable transformants, 1×10⁷ U937T cells were washed in RPMI-1640 medium and resuspended in 0.2 ml of Isceve’s modified Dulbecco’s medium (Sigma-Aldrich) without FBS. A total of 20 μg plasmid in 20 ml double-distilled H₂O was transferred to an electroporation cuvette with a 0.4-cm gap (Bio-Rad Laboratories, Hercules, CA, USA). Electroporation was performed using a Gene Pulser II Electroporation system (Bio-Rad Laboratories) at 170 V and 960 mF. The samples were then transferred to complete RPMI-1640 medium. Subsequent to a 24-h resting period, 1 mg/ml tetracycline, 0.5 mg/ml puromycin and 500 mg/ml hygromycin B (Clontech Laboratories, Inc.) were added and cells were incubated at 37°C in 5% CO₂. The positive polyclonal population (pool) was identified based on the induction of the corresponding protein expression following tetracycline withdrawal, and were named U937^ANP32B(WT), U937^{ANP32B(D163A)} and U937^{ANP32B(1-163)}. An empty plasmid-transfected cell line called U937^empty was used as the control. All cells were maintained in RPMI-1640 medium supplemented with 10% FBS, 1 mg/ml tetracycline, 0.5 mg/ml puromycin and 0.5 mg/ml hygromycin B.

To induce apoptosis, ~3×10⁵ cells/ml were initially seeded and cultured in tetracycline-free Isceve’s modified Dulbecco’s medium for 10 days to induce the expression of the corresponding proteins. The cells were then incubated with 1 μM etoposide (Enzo Life Sciences, Inc., Farmingdale, NY, USA) for an additional 12 h. The annexin-V assay was performed using a flow cytometer (BD FACSCanto II; BD Biosciences, San Jose, CA, USA) according to the manufacturer’s instructions for the ApoAlert Annexin-V Apoptosis kit (Clontech Laboratories, Inc.), in order to count the number of annexin-V-positive apoptotic cells. Caspase-3 activity was determined by the Caspase 3 Assay kit, Colorimetric (Sigma-Aldrich).

The protein lysates were equally loaded onto a 10–12% SDS-PAGE gel using a mini gel apparatus (Bio-Rad Laboratories, Inc.) and subsequently transferred to a nitrocellulose membrane (Bio-Rad Laboratories). The membranes were blocked with 5% nonfat dry milk solution in tris-buffered saline (Beyotime) for 1 h at room temperature and then incubated in primary antibody dissolved in the 5% nonfat dry milk solution at 4°C overnight. The following antibodies were used: Polyclonal rabbit anti-human protein kinase Cδ (PKCδ) (C-20; SC-937; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA); polyclonal rabbit anti-human cleaved caspase-3 (9661; Cell Signaling Technology, Danvers, MA, USA); monoclonal mouse anti-human poly[ADP (adenosine diphosphate)-ribose] polymerase (PARP; F-2; SC-8007; Santa Cruz Biotechnology, Inc.); polyclonal rabbit anti-human Bcl-2 (sc-492; Santa Cruz Biotechnology, Inc.) and polyclonal rabbit anti-human ANP32B (10843-1-AP; ProteinTech Group, Inc., Chicago, IL, USA), with monoclonal mouse anti-β-actin mAb (EMD Millipore, Billerica, MA, USA) to confirm equal loading. Following washing with PBS, the blots were incubated with horseradish peroxidase-conjugated secondary antibody (Dako, Glostrup, Denmark) corresponding to the primary antibody in 5% nonfat dry milk solution for 1 h at room temperature. Proteins were then detected using a luminol detection reagent (Santa Cruz Biotechnology, Inc.), and developed onto X-ray films (Kodak, Rochester, NY, USA).

Cells were collected onto slides using a Shandon Cytospin 4 Cytocentrifuge (Thermo Fisher Scientific, Waltham, MA, USA) at 800 × g for 5 min and fixed with 4% formaldehyde (Beyotime) for 10 min. Subsequent to permeabilization with 0.3% Triton X-100 (Beyotime) in phosphate-buffered saline (PBS; Beyotime) for 10 min, cells were incubated with 1% bovine serum albumin (Beyotime) in PBS for 2 h at room temperature, followed by overnight incubation with the ANP32B antibody. Cells were stained with fluorescein isothiocyanate-labeled anti-rabbit IgG (Dako) for 1 h. Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (Molecular Probes Life Technologies, Eugene, OR, USA). Fluorescent signals were measured using an MRC-1024 laser scanning confocal microscope equipped with a Zeiss ×60 objective (Bio-Rad Laboratories) or an Olympus BX51 fluorescence microscope (100x/1.30 oil objective lens; Olympus Corporation, Tokyo, Japan).

The differences between the two groups were analyzed for statistical significance using the nonparametric Wilcoxon rank sum test. P<0.05 was considered to indicate a statistically significant difference.

In previous studies, ANP32B has been demonstrated to be a caspase-3 substrate during apoptotic induction, and is primarily cleaved after Asp163 (12). To determine the functional significances of ANP32B cleavage during apoptosis, leukemic U937T cell lines with inducible expression of WT, D163A mutant and N-terminal fragment (1-163) ANP32B, were generated by a Tet-Off gene expression system. In the Tet-off system, gene expression is turned on when tetracycline is removed from the culture medium. U937T^empty, U937T^ANP32B(WT), U937T^{ANP32B(D163A)} and U937T^{ANP32B(1-163)} cells were all induced to express flag-tagged ANP32B or its mutants, by tetracycline withdrawal for 6–12 days (Fig. 1). Immunofluorescent analysis with the antibody against ANP32B indicated that ANP32B(WT) and ANP32B(D163A) proteins were localized in the nuclei, while ANP32B(1-163) was in the cytosol (Fig. 2). This was due to the absence of the nuclear localization signal (NLS), which was mapped to amino acid residues KRKR at position 239–242 in the C-terminal of this protein.

The contribution of caspase-3-mediated cleavage of ANP32B to apoptosis was investigated by inducing the above cell lines to undergo apoptosis and measuring the number of annexin-V-positive cells using flow cytometry (Fig. 3A). The results demonstrated that compared with U937T^empty cells (18.3±1.9%), apoptosis was significantly increased in U937T^ANP32B(WT) cells (37.2±2.7%; P<0.05) as well as highly significantly enhanced in U937T^{ANP32B(D163A)} cells (58.5±2.1%; P<0.01), but was not markedly altered in U937T^{ANP32B(1-163)} cells (21.8±0.8%) (Fig. 3B). This suggested that the uncleavable ANP32B(D163A) possesses pro-apoptotic activity.

The catalytic activity of PKCδ is established to be important at the early stage of etoposide-induced apoptosis (prior to caspase-3 activation) (24,25), while activated caspase-3 produces an amplifying effect on PKCδ activation (26). To further confirm the roles of different ANP32B forms in apoptosis, the proteolytic activation of PKCδ and caspase-3 was investigated, in addition to the cleavage of PARP, a common substrate of activated caspase-3 during apoptosis. As depicted in Fig. 4A, treatment with 1 μM etoposide induced slight fragmentation of PKCδ, caspase-3 and PARP in U937T^empty cells, while these fragmentations were moderately and significantly enhanced in U937T^ANP32B(WT) and U937T^{ANP32B(D163A)} cells, respectively, but remained unchanged in U937T^{ANP32B(1-163)} cells. Caspase-3 activation was observed to be regulated similarly (Fig. 4B).

ANP32B has been reported to regulate gene transcription as a histone chaperone in the nucleus (27), thus, the mechanisms underlying the pro-apoptotic role of full-length ANP32B were investigated, via the detection of several key apoptosis-associated genes, including Bcl-2, Mcl-1 and inhibitor of caspase-activated DNase (ICAD) in the inducible cell lines. The data indicated that the expression levels of anti-apoptotic Bcl-2 protein were progressively reduced subsequent to tetracycline removal, in parallel with the induction of full-length ANP32B protein in U937T^ANP32B(WT) and U937T^{ANP32B(D163A)} cells, while Mcl-1 and ICAD expression remained unchanged (Fig. 5). In addition, the expression of Bcl-2 was not affected by ANP32B(1-163), which was localized in the cytosol and had no effect on apoptosis induction. The reduced expression of Bcl-2 protein is hypothesized to contribute to the apoptosis-enhancing ability of full-length ANP32B.