WNT974 antagonizes the Wnt signaling pathway in human CML stem/progenitor cells. (A) Wnt secretion was evaluated in WNT1-MSC cultured in the presence of WNT974 for 24 h. Conditioned medium was harvested and added onto 293T-BAR reporter cells. WNT-β-catenin transcriptional activity was then evaluated after a further 24 h (n = 5). (B) HEK 293T cells overexpressing Wnt-1 were treated with WNT974, metabolically labeled with azide-containing palmitic acid and modified palmitoylated proteins detected by labeling with alkyne-containing APC dye using flow cytometry (n = 4). (C) CML CD34⁺ cells were cultured in the presence or absence of human MSC in the presence of WNT974 for 48 h, and immunofluorescence microscopy was performed. CML CD34⁺ cells labeled with antibodies to β-catenin (green) and DAPI (blue) are shown. Two samples were studied. All scale bars represent a size of 10 µM, and at least 200 cells were analyzed for each sample. (D) qPCR analysis for mRNA expression of Wnt target genes in CML CD34⁺ cells (n = 5) cultured as in panel A. Error bars represent mean ± SEM. Ctrl, control; ns, not significant; PPAR, peroxisome proliferator-activated receptors. *P < .05; **P < .01; ***P < .001; ****P < .0001.