*P?0.05, **P?0.01 compared to controls. Discussion NB is the most common malignancy of infancy and constitutes 50% of all infantile GS-9451 cancers. colony forming ability, cell apoptosis and the percentage of different cell cycle. The Western blot was used for detecting the expression of key proteins of Wnt/ beta-catenin (Wnt/-catenin) signaling pathway. Results The results showed that TNKS1 inhibition decreased the viability of SH-SY5Y, SK-N-SH and IMR-32 cells, induced apoptosis in SH-SY5Y as well as SK-N-SH cells, and led to the accumulation of NB cells in the S and G2/M phase of the cell cycle. Moreover, we demonstrated TNKS1 inhibition may in part blocked Wnt/-catenin signaling and reduced the expression of anti-apoptosis protein. Finally, we also demonstrated that TNKS1 inhibition decreased colony formation in vitro. Conclusions These findings suggested that TNKS1 may be a potential molecule target for the treatment of NB. demonstrated that XAV939 or siRNA-mediated abrogation of TNKS expression increases Axin1 and Axin2 protein levels and attenuates Wnt-induced transcriptional responses in several breast cancer lines . In our previous studies, we have known that TNKS1 was also up-regulated in NB SH-SY5Y cells (data not shown). It has also been reported that the -catenin has a close relationship with the prognosis of NB. The stronger the -catenin expressed in nucleus, the GS-9451 higher risk of NB would be, and the worse the prognosis was . However, whether the proliferation of NB cell lines could be inhibited through blocking the Wnt pathway or other mechanisms? In the present study, we have investigated the anti-proliferative effect of Ctsd XAV939 on the human NB cell lines. In addition, we studied the cell apoptosis induced by XAV939 and assessed the role of Wnt signaling in it. Materials and methods Cell culture and TNKS1 inhibitor Human NB SH-SY5Y, SK-N-SH and IMR-32 cells were obtained from the American Type Culture Collection (ATCC; Rockville, USA). Cells were maintained in Dulbeccos modified Eagles medium (DMEM; GS-9451 Hyclone), with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin (Sigma Chemical Co., St Louis, Missouri) and were grown in a 5% CO2 incubator at 37C. The TNKS1 inhibitor XAV939 was purchased from Sigma Aldrich. Assessment of cellular viability Cellular viability was assessed by MTT method. GS-9451 Briefly, equal numbers of NB SH-SY5Y, SK-N-SH and IMR-32 cells were plated at a density of 1 1??104 per well in 96-well plates, and were treated with various concentrations of XAV939 for 24, 48, or 72?h. 20?l MTT (5?mg/ml) were incubated with cells of each sample for 4?h, then were replaced with 150? l DMSO and 96-well plates were rotated gently for 10?min. Cell viability was determined by measuring colorimetric absorbance at 490?nm, and was read with a microplate reader . Experiments were done in triplicate and average activity rates relative to control and standard errors were calculated. Colony formation assay Colony formation assays were performed as described . Briefly, SH-SY5Y cells were plated in triplicate at 100 cells per well in 6-well plates and cultured in DMEM medium supplemented with 10% FBS. After 4-5?h, cells were treated with DMSO or XAV939, as well as transfected with lentivirus-mediated scrambled-shRNA (SCR group) or TNKS1-shRNA (shRNA group). Colonies were allowed to form for 14?days and fixed in methanol for 15?minutes, and dyed with crystal violet for 15?minutes at room temperature. Afterward, the dye was washed off and colonies that contained more than 50 cells were counted. The colony formation efficiency was the ratio of the colony number to the planted cell number. Apoptosis assays Apoptosis was measured using Annexin V/FITC Apoptosis Detection kit (KeyGEN Biotech, Nanjing, China) following the manufacturers protocol. Briefly, equal numbers of SH-SY5Y and SK-N-SH cells, treated with DMSO or XAV939 for 24, 48, or 72 h, were incubated with Annexin V-FITC, followed by staining of their DNA with propidium iodide (PI) in the dark. Then, each sample was analyzed by fluorescence-activated cell sorting (FACS) (BD, San Jose, CA, USA). The percentages of cells staining positive for Annexin V were calculated, and means GS-9451 as well as standard error were plotted. Alternatively, apoptosis was also determined using Hoechst 33342 staining. After treatment, cells were washed with PBS and stained with Hoechst 33342 (10 g/mL, Sigma Aldrich). Then the cells were observed by fluorescent microscope (Olympus Inverted.