Identification of the canonical Wnt pathway as major downstream signaling of TIM-3/Gal-9 autocrine loop. (A) Fluorescence-activated cell sorting (FACS) analysis of surface TIM-3 expression (left) and intracellular Gal-9 expression (right) in KASUMI-3 cells (B) Percentage of BM chimerism in NSG mice with xenografts with TIM-3 KD KASUMI-3 cells (n = 6) and scrambled control cells (n = 6) (left panel), and human Gal-9 concentration in serum of mice with xenografts (right panel). ∗∗P < .01 vs scrambled control. These data were obtained at 13 weeks after xenotransplantation. (C-D) Enrichment plots for gene sets significantly enriched in scrambled control cells compared with KASUMI-3 cells transfected with shHAVCR2-1 and shHAVCR2-2. (C) HSC- and LSC-related genes.³⁵ (D) Canonical Wnt pathway–related genes (BIOCARTA). (E) Quantification of nucleus translocation of β-catenin evaluated by ArrayScan system. Left panels show the representative images for localization of β-catenin in scrambled control and TIM-3 KD KASUMI-3 cells. Scale bar represents 10 μm. Right panel shows the quantification of β-catenin (green) translocation to the nucleus calculated from the fluorescence intensity and area overlapped with nucleus (blue), which were analyzed in TIM-3 KD KASUMI-3 cells and scrambled control cells. Data are presented as mean ± SEM, ∗∗P < .01 vs scrambled control.