The human neuroblastoma cell lines SK-N-AS, BE(2)C, SK-N-DZ, SK-N-F1 and SHEP1 were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). The BE(2)C cells were cultured in a 1:1 mixture of Dulbecco's modified Eagle's medium (DMEM) and Ham's nutrient mixture F12 (DMEM/F12; Thermo Fisher Scientific, Inc., Waltham, MA, USA) with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin (P/S). SK-N-AS, SK-N-DZ, SK-N-F1 and SHEP1 cells were cultured in DMEM (Thermo Fisher Scientific, Inc.) with 10% FBS and 1% P/S. The 293FT cell line (ATCC) was cultured with DMEM (Gibco; Thermo Fisher Scientific, Inc.) containing 1% glutamine, glycine and pyruvate. All cells were incubated at 37°C in an incubator with 5% CO₂.

TP53BP2 short hairpin (shRNA) and green fluorescent protein (GFP) shRNA were purchased from BGI (Shanghai, China). The sequences of TP53BP2 shRNA (shTP53BP2) were as follows: shTP53BP2-1# forward, 5′-CACCGCAGAATGCCAAGCTACAACACGAATGTTGTAGCTTGGCATTCTGC-3′ and reverse, 5′-AAAAGCAGAATGCCAAGCTACAACATTCGTGTTGTAGCTTGGCATTCTGC-3′; and shTP53BP2-2# forward, 5′-CACCGCTGCAGTAGGTCCCTATATCCGAAGATATAGGGACCTACTGCAGC-3′ and reverse, 5′-AAAAGCTGCAGTAGGTCCCTATATCTTCGGATATAGGGACCTACTGCAGC-3′. The PLKO.1 vector (Thermo Fisher Scientific, Inc.) was digested using AgeI and BamHI, and the annealed human TP53BP2 shRNA was inserted into the PLKO.1 vector using T4 ligase. Subsequently, the vector was transformed and monoclonal clones were selected for sequencing. The plasmids were extracted using a plasmid extracted kit (Tiangen Biotech Co., Ltd., Beijing, China). Lentiviruses were generated by co-transfecting the 293FT cell line with the packaging plasmids pLP1, pLP2 and pLP/VSVG (all from Invitrogen; Thermo Fisher Scientific, Inc.) and the shRNA plasmids. Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) was used for all transfections, according to the manufacturer's protocol. After 48 h, virus-containing media were harvested. BE(2)C and SK-N-DZ cells were plated at a density of 5×10⁵ cells in 100 mm plates and cultured for 24 h. Subsequently, the virus-containing media were mixed with 4 µg/ml Polybrene (Invitrogen; Thermo Fisher Scientific, Inc.) and used for cell infection. At 24 h after infection, the medium was removed and cells were cultured with 2 mg/ml puromycin for 2 days. The cells were then selected for subsequent experiments.

MTT assays were used to investigate cell proliferation. Briefly, ~1,000 cells were seeded in 96-well plates with 200 µl DMEM/F12. After 24 h of culture, 20 µl MTT was added to each well and the cells were incubated for 4 h. Dimethylsulfoxide was added to the wells to dissolve the purple formazan crystals. After 10 min on a shaking table, the absorbance was determined at 560 nm using a microplate reader daily for 7 days. All experiments were performed independently in triplicate.

For BrdU immunofluorescence staining, BE(2)C and SK-N-DZ cells were cultured in 24-well plates and incubated with 10 µg/ml BrdU for 45 min at 37°C. The cells were then washed with PBS three times and fixed for 20 min with 4% paraformaldehyde (PFA) at room temperature. Subsequently, the cells were exposed to 0.3% Triton X-100 for 5 min, treated with 1 mol/l HCl and blocked for 1 h with 10% goat serum (Thermo Fisher Scientific, Inc.) diluted with PBS at room temperature. The cells were then incubated with a monoclonal rat primary antibody against BrdU (1:200; catalog no. ab6326; Abcam, Cambridge, UK) for 1 h at room temperature and with Alexa Fluor^® 594 goat anti-rat immunoglobulin G (IgG) secondary antibody (1:400; catalog no. A-21211; Invitrogen; Thermo Fisher Scientific, Inc.) for 1 h at room temperature. DAPI (300 nM) was used for nuclear staining for 15 min at room temperature. A Nikon 80i fluorescence microscope (Nikon Corporation, Tokyo, Japan) was used to observe the number of stained cells (magnification, ×200). Images of ten randomly selected microscopic fields were captured.

Soft agar assays were used to determine cell tumorigenicity. In total, 800 BE(2)C and SK-N-DZ cells were mixed with 0.3% Noble agar in DMEM/F12 and then plated in 6-well plates containing a solidified bottom layer of 0.6% Noble agar in growth medium. Following solidification, the plates were transferred to a 37°C incubator with 5% CO₂. After 4 weeks, colonies were stained with MTT (1 µg/ml) for 30 min at 37°C and images were captured using a light microscope (magnification, ×200).

RT-qPCR was used to determine the expression of mRNA. According to the manufacturer's protocol, 1 ml TRIzol^® (Takara Bio, Inc., Otsu, Japan) was added to each group (shGFP-, shTP53BP2-1#- and shTP53BP2-2#-transfected cells, and untransfected cells), and the cells were then harvested and extracted for total RNA purification. The mRNA was reverse-transcribed into cDNA using Moloney murine leukemia virus reverse transcriptase (Promega Corporation, Madison, WI, USA). The mRNA expression levels of TP53BP2, ATG3, ATG5 and ATG7 were determined using the SYBR Green PCR Master mix (Takara Bio, Inc.) using qPCR. The qPCRs were performed in triplicate using the OneStep plus7500 RT-PCR system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The amplification conditions were as follows: 95°C for 10 min, 95°C for 15 sec, 60°C for 30 sec and 35 cycles of 30 sec at 72°C. The following primer sequences were used: GAPDH forward, 5′-ACGGATTTGGTCGTATTGGG-3′ and reverse, 5′-TCCTGGAAGATGGTGATGGG-3′; TP53BP2 forward, 5′-AGTCAGTTCCTTGTGGAGCC-3′ and reverse, 5′-CCGCAGAAACACCTGTGAAC-3′; ATG3 forward, 5′-TTGGCTATGATGAGCAACGG-3′ and reverse, 5′-CCCCTCCTTCTGCAACAGTCT-3′; ATG5 forward, 5′-TCAGCTCTTCCTTGGAACATCA-3′ and reverse, 5′-CCCATCCAGAGTTGCTTGTGA-3′; and ATG7 forward, 5′-TTTGCTTCCGTGACCGTACC-3′ and reverse, 5′-CTTTTCCCATCCAACTGCTTTA-3′. The data were analysed using the 2^−ΔΔCq method (27). GAPDH was used as the control.

SK-N-AS, BE(2)C, SK-N-DZ, SK-N-F1 and SHEP1 cells were digested with trypsin and washed twice with PBS. Cells were harvested and suspended in 1% radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Haimen, China). Protein concentrations were measured using a Bicinchoninic Acid protein assay kit (Beyotime Institute of Biotechnology). Total protein (50 µg) was separated using SDS-PAGE (30% gel) and then the proteins were transferred onto polyvinylidene difluoride membranes. Following blocking for 2 h with 5% goat serum (diluted with PBS) at room temperature, the membranes were incubated overnight at 4°C with the following primary antibodies: Anti-TP53BP2 (1:1,000, catalog no. ab181377; Abcam), anti-LC3 II (1:1,000; catalog no. ab48394; Abcam) and anti-tubulin (1:5,000; catalog no. ab7291; Abcam). The membranes were than incubated at room temperature for 2 h with horseradish peroxidase-conjugated anti-mouse IgG secondary antibody (1:10,000; catalog no. 04-18-06) or horseradish peroxidase-conjugated rabbit anti-goat IgG secondary antibody (1:10,000; catalog no. 14-13-06; both from KPL, Inc., Gaithersburg, MD, USA). Proteins were visualized using BeyoECL Plus reagent (Beyotime Institute of Biotechnology). Western blot data were quantified with Gel-Pro Analyzer 4 software (Media Cybernetics, Inc., Rockville, MD, USA).

The expression of LC3 II was detected using immunofluorescence. In total, ~2×10⁴ cells were seeded into 24-well plates, washed three times with PBS and fixed for 20 min with 4% PFA at room temperature. PBS with 1% Triton X-100 was then added for 20 min. Following three washes with PBS, the cells were blocked for 1 h with 4% goat serum (diluted with PBS) at room temperature. The cells were then incubated with a rabbit monoclonal antibody against LC3 II (1:100; catalog no. ab48394; Abcam) at 4°C overnight. Alexa Fluor^® 488 goat anti-rat IgG secondary antibody (1:400; Invitrogen; catalog no. O-6382; Thermo Fisher Scientific, Inc.) was then added for 2 h at room temperature in the dark. DAPI (300 nM) was used for nuclear staining for 15 min at room temperature. Images of ten randomly selected microscopic fields were captured using a confocal microscope (magnification, ×2,000).

Patient data and gene expression datasets were obtained from the R2: Microarray analysis and visualization platform (http://hgserver1.amc.nl/cgi-bin/r2/main.cgi), which contains data from the ‘Tumour Neuroblastoma public Versteeg’, ‘Tumour Neuroblastoma-SEQC’ and ‘Tumour Neuroblastoma-Kocak’ datasets (28). These datasets contain mRNA expression data and no protein expression data. The Versteeg dataset contains 88 cases of neuroblastoma with tumor grade and gene variation. All prognosis analyses were performed with R2, and all data and P-values from a log-rank test were downloaded. Kaplan-Meier analysis was performed and the resulting survival curves were generated using GraphPad Prism 6.0 software (GraphPad Software, Inc., La Jolla, CA, USA). All cut-off values for generating high and low expression groups were determined using the online R2 database algorithm.

All data were analyzed using SPSS software (version 20.0; IBM Corp., Armonk, NY, USA). Quantitative data are presented as the mean ± standard deviation. One-way analysis of variance followed by Fisher's least significant difference test was used to assess significant differences. P<0.05 was considered to indicate a statistically significant difference.

To investigate whether TP53BP2 can be used as a prognostic indicator of neuroblastoma. A Kaplan-Meier analysis of progression-free survival for the Versteeg data indicated that an increased expression level of TP53BP2 is associated with a poor prognosis and a low expression level of TP53BP2 is associated with improved overall survival (Fig. 1A). Similar results were observed for the SEQC and Kocak data (Fig. 1B and C). In summary, all data indicated that an increased expression level of TP53BP2 is associated with a poor prognosis for patients with neuroblastoma.

Subsequently, RT-qPCR and western blot assays were performed to determine the expression of TP53BP2 in the neuroblastoma cell lines SK-N-AS, BE(2)-C, SK-N-DZ, SK-N-F1 and SHEP1. It was identified that TP53BP2 was expressed in all five cell lines (Fig. 1D and E). The aim of these experiments was to illustrate the involvement of TP53BP2 in the tumorigenesis of neuroblastoma (29).

To investigate the role of TP53BP2 in cell proliferation, the human neuroblastoma cell lines BE(2)C and SK-N-DZ were selected. Lentivirus vectors containing shTP53BP2 were successfully constructed and shGFP was used as a control (30,31). Through screening, stable TP53BP2-knockdown cells were established. Western blot and RT-qPCR analysis revealed that TP53BP2 was significantly downregulated in the transfected cells compared with in the controls (Fig. 2A-D). Subsequently, immunofluorescence staining using a BrdU label in BE(2)C and SK-N-DZ cells confirmed that cell proliferation was significantly inhibited in the TP53BP2-knockdown cells compared with in the controls for the two cell lines (Fig. 3). Furthermore, MTT assays demonstrated that downregulation of TP53BP2 significantly decreased cell proliferation (Fig. 4A and B). These results indicate that TP53BP2 is essential for the proliferation of neuroblastoma cells.

In numerous studies, a soft agar assay has been used as a human tumor stem-cell assay to investigate the ability of individual cancer cells to proliferate and form colonies (32). As presented in Fig. 4C and D, the role of TP53BP2 in neuroblastoma tumorigenesis was examined, which revealed that the colonies were smaller and significantly fewer in number for the TP53BP2-knockdown cells compared with for the controls (Fig. 4C and D). These results indicate that TP53BP2 can suppress neuroblastoma cell colony formation in vitro.

Immunofluorescence assays were performed to detect the expression of LC3 II, which is a marker of autophagy (33). The results revealed that LC3 II expression increased markedly in the TP53BP2-knockdown cells compared with the controls (Fig. 5A). Furthermore, the expression levels of LC3B proteins in TP53BP2-knockdown and shGFP neuroblastoma cells were detected using western blot analysis. It was identified that the expression level of LC3 II was significantly increased in the TP53BP2-knockdown cells compared with in the controls (Fig. 5B-G). To confirm the occurrence of autophagy, RT-qPCR analysis revealed that the expression levels of ATG3, ATG5 and ATG7 were significantly upregulated in TP53BP2-knockdown cells (Fig. 5H and I). These results indicate that the inhibition of TP53BP2 upregulates the expression of LC3 II and ATG proteins. This suggests that the downregulation of TP53BP2 promotes the upregulation of LC3 II and induces autophagy.