HCK is a critical signal transducing molecule involved in TIM-3–induced canonical Wnt pathway activation. (A) WB analysis of Gal-9–stimulated KASUMI-6 cells in the presence of SFK inhibitors: AMGT, PP1, PP2, and A-419259 (potent HCK inhibitor). Inhibitable members of SFKs at each concentration of inhibitors are listed. Because KASUMI-6 cells showed obvious response to SFKs inhibitors, we presented the representative data of KASUMI-6 cells. (B) WB analysis of cell lysates from Gal-9–stimulated TIM-3⁺ primary samples in the presence of 0, 1, and 10 nM of A-419259. Cells were preincubated with A-419259 for 2 hours before stimulation with Gal-9. Representative results out of 4 independent TIM-3⁺ AML cases (AML#6, #9, #14, and #17) are shown. (C) Images of KASUMI-3 cells captured from representative fields obtained by ArrayScan analysis. These cells were stimulated with or without Gal-9 in the presence or absence of A-419259 for 20 hours. Scale bar represents 10 μm. (D) Extent of β-catenin translocation to the nucleus evaluated using the ArrayScan system compared with nonstimulated control. Data are presented as mean ± SEM, ∗P < .05 and ∗∗P < .01. (E) Immunoblotting analysis with HCK, pSFK, and TIM-3 antibodies of total cell lysates (left) and immunoprecipitated lysates (right). Cells were stimulated with Gal-9 for 0, 5, and 10 minutes and subsequently lysed and subjected to IP with an HCK antibody or normal rabbit IgG. Representative results (AML#13) out of 3 independent experiments are shown here.