Figure 2.
Gal-9 ligation to TIM-3 induces LRP6 signalosome formation and subsequent β-catenin accumulation in AML. (A) WB analysis of β-catenin accumulation induced by stimulation with Gal-9 in the presence of inhibitors in KASUMI-3 cells. KASUMI-3 cells were stimulated with Gal-9 in the presence of dimethyl sulfoxide, 10 μM of U0126 (MEK1/2 inhibitor), 10 μM of LY294002 (PI3K inhibitor), or 200 ng/mL of DKK-1, and their total cell lysates subjected to WB analysis. (B) Summarized data of Gal-9 stimulation–induced changes in the accumulation of β-catenin from 3 independent experiments. (C) Extent of β-catenin translocation to the nucleus evaluated using the ArrayScan system compared with that observed in nonstimulated controls. Data in panels B and C are presented as mean ± SEM, ∗P < .05. (D) Immunoblotting analysis of total lysates (left) and IP lysates (right) of Gal-9–stimulated primary TIM-3+ AML samples. Cells were lysed and subjected to IP with an anti-LRP6 antibody or normal mouse IgG. A representative result of TIM-3+ primary AML cells out of 3 independent cases (AML#4, #9, and #13) is shown. (E) WB analysis of Gal-9 stimulation–induced phosphorylation of LRP6 at Thr1479 in mock-transfected and TIM-3-OX THP-1 cells. (F) WB analysis of cell lysates from TIM-3+ AML cells (left) and TIM-3− AML cells (right). TIM-3+ AML cells were stimulated with Gal-9 in the absence or presence of DKK-1 (200 ng/mL). Representative results out of 4 independent TIM-3+ AML cells (AML#1, #2, #4, and #16) and 2 TIM-3− AML cells (AML#10 and #11) are shown. n.s., no significant difference.

Gal-9 ligation to TIM-3 induces LRP6 signalosome formation and subsequent β-catenin accumulation in AML. (A) WB analysis of β-catenin accumulation induced by stimulation with Gal-9 in the presence of inhibitors in KASUMI-3 cells. KASUMI-3 cells were stimulated with Gal-9 in the presence of dimethyl sulfoxide, 10 μM of U0126 (MEK1/2 inhibitor), 10 μM of LY294002 (PI3K inhibitor), or 200 ng/mL of DKK-1, and their total cell lysates subjected to WB analysis. (B) Summarized data of Gal-9 stimulation–induced changes in the accumulation of β-catenin from 3 independent experiments. (C) Extent of β-catenin translocation to the nucleus evaluated using the ArrayScan system compared with that observed in nonstimulated controls. Data in panels B and C are presented as mean ± SEM, ∗P < .05. (D) Immunoblotting analysis of total lysates (left) and IP lysates (right) of Gal-9–stimulated primary TIM-3+ AML samples. Cells were lysed and subjected to IP with an anti-LRP6 antibody or normal mouse IgG. A representative result of TIM-3+ primary AML cells out of 3 independent cases (AML#4, #9, and #13) is shown. (E) WB analysis of Gal-9 stimulation–induced phosphorylation of LRP6 at Thr1479 in mock-transfected and TIM-3-OX THP-1 cells. (F) WB analysis of cell lysates from TIM-3+ AML cells (left) and TIM-3− AML cells (right). TIM-3+ AML cells were stimulated with Gal-9 in the absence or presence of DKK-1 (200 ng/mL). Representative results out of 4 independent TIM-3+ AML cells (AML#1, #2, #4, and #16) and 2 TIM-3− AML cells (AML#10 and #11) are shown. n.s., no significant difference.

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