Enhanced Wnt–β-catenin signaling in TKI-treated CML stem/progenitor cells cocultured with MSCs. (A) Western blotting for P-Crkl, N-cadherin, β-catenin, P-GSK3β (S9), and actin in CML CD34⁺ cells cultured with and without MSCs and with and without IM treatment. (B) Western blotting for N-cadherin, β-catenin, P-GSK3β (S9), and actin in CML CD34⁺ cells and MSC-adherent and MSC-nonadherent CML CD34⁺ cells with and without IM treatment, and in CML CD34⁺ cells cocultured with MSCs and IM and NCDH or control peptide as shown. (C) Immunofluorescence microscopy of CML CD34⁺ cells labeled with antibodies to N-cadherin (green) and β-catenin (red) following culture as shown. Nuclei were labeled with 4,6 diamidino-2-phenylindole (DAPI; blue). Results shown are representative of 100 cells analyzed per slide. (D) N-cadherin and β-catenin protein–protein interactions were evaluated by using Duolink in situ PLA technology. Protein interactions are visualized as red dots. (E) Cytoplasm (cyt) and nuclear (nucl) fractions from CML CD34⁺ cells cultured with and without MSCs were analyzed by western blotting for β-catenin, α-tubulin, and sp1. (F) Wnt–β-catenin–related transcriptional activity was evaluated by using an improved TOPFlash reporter system in CML CD34⁺ cells cultured as shown. (G) Q-PCR analysis for mRNA expression of the Wnt–β-catenin target genes cyclin-D1, c-Myc, and peroxisome proliferator-activated receptor delta (PPARD) in CML CD34⁺ cells cultured as shown. (H) Western blotting for β-catenin, N-cadherin, P-GSK3β (S9), and actin in normal CD34⁺ cells cultured with and without MSCs. (I) Q-PCR analysis for mRNA expression of N-cadherin, β-catenin and Wnt–β-catenin target genes cyclin-D1, c-Myc, and PPARD in normal CD34⁺ cells cultured as shown. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; ns, not significant. n = 3. *P < .05; **P < .01; ***P < .001.