Figure 4. JAM3 collaborates with LRP5 to activate β-catenin/CCND1 signaling.

(A) Phospho–β-catenin (S552) and total β-catenin levels were evaluated between WT and Jam3-null YFP⁺Mac-1⁺c-Kit⁺ LICs by immunoblotting. (B) β-Catenin levels were compared between WT and Jam3-null YFP⁺Mac-1⁺c-Kit⁺ LICs by immunofluorescence staining. Scale bars: 5 µm. (C) A constitutively active form of phospho–β-catenin (S37A, β-catenin^CN) was subcloned in the pCDH-EF1a-T2A-mCherry vector and ectopically expressed in Jam3-null leukemia cells, which were then injected into recipient mice. Survival was compared among the mice receiving WT cells, Jam3-null cells, and β-catenin^CN–overexpressing WT or Jam3-null cells (n = 5–6; *P < 0.05, **P < 0.01, log-rank test). (D) Phospho–β-catenin (S552) and total β-catenin levels were validated in leukemia cells from the rescue experiment in C. (E) The cell cycle distribution in YFP⁺Mac-1⁺c-Kit⁺ LICs from the rescue experiment in C was determined using Ki-67 and Hoechst 33342 staining (n = 3; ***P < 0.001, 2-way ANOVA followed by Bonferroni’s post-test). (F) StrepII-tagged JAM3 and FLAG-tagged LRP5 were overexpressed in 293T cells, and their lysates were coimmunoprecipitated by strepII beads, followed by Western blotting analysis for FLAG (LRP5). (G) A reverse coimmunoprecipitation experiment was performed after LRP5-FLAG pull-down, followed by Western blotting analysis for strepII (JAM3). The empty vector was used as the control. Experiments were conducted 3 times for validation.