Figure 6

Tenovin-6 suppresses Wnt/β-catenin signaling in ALL cells and eliminates ALL stem/progenitor cells. A, REH and NALM-6 cells were treated with various concentrations of Tenovin-6 for 24 and 48 hours. Expression of β-catenin, c-myc, and cyclin D1 was detected by Western blotting with the indicated antibodies. B, REH and NALM-6 cells were treated with increasing concentrations of Tenovin-6 for 48 hours, and then qRT-PCR analysis of β-catenin target genes (c-Myc, cyclin D1, and LEF1) was performed using intron-spanning primers. C, REH and NALM-6 cells were treated with various concentration of Tenovin-6 for 48 hours. Western blotting for Dvl3 in total cell lysate and β-catenin in nuclear extract was performed. PCNA was used as a nuclear protein reference. D, EMSA. Nuclear fractions from REH and NALM-6 cells treated with increasing concentration of Tenovin-6 for 48 hours were incubated with wild-type biotin-labeled TCF probe. Cold competition assay was done using 100-fold unlabeled wild-type probe. E, CD133 MicroBeads separating system was applied to bone marrow mononuclear cells of 3 patients with ALL. The sorted CD133⁺ cells were then untreated or treated with 5 μM Tenovin-6 for 48 hours, and apoptosis was evaluated by flow cytometry after cells were stained with Annexin V-FITC and CD19-phycoerythrin.