SALL4 and the Wnt/β-catenin signaling pathway. (A) Both SALL4A and SALL4B can interact with β-catenin. Nuclear extracts (lysates) prepared from Cos-7 cells were transiently transfected with HA-SALL4A or HA-SALL4B. (i) Anti-HA antibody recognized both SALL4A (165 kDa) and SALL4B (95 kDa). (ii) β-Catenin was detected in the lysates. (iii) Immunoprecipitation was performed with the use of an HA affinity resin and detected with an anti-β-catenin antibody. β-Catenin was readily detected in both HA-SALL4A and HA-SALL4B pull-downs. Untransfected cells subjected to the same immunoprecipitation condition as the transfected cells were used as a control. (B) Activation of the Wnt/β-catenin signaling pathway by both SALL4A and SALL4B. HEK-293 cells were transfected with 1.0 μg of either mock alone, or SALL4A or SALL4B plasmid, with or without Wnt1 (including Wnt1, and its coactivators: LRP6, MESD, and F₂5), and TOPflash (TOP) or FOPflash (FOP) reporter plasmid (Upstate USA, Chicago, IL). After 24 hours, luciferase activity was measured. SALL4A or SALL4B alone showed more potent activation of Wnt signaling pathway when compared with the positive control Wnt1. In addition, both SALL4 isoforms demonstrated a significantly synergistic activation of the Wnt signaling pathway with Wnt1. Data represent mean ± SD of 3 independent experiments. (C) Up-regulation of c-Myc and Cyclin D1 expression in SALL4B transgenic mice. RT-PCR analysis was performed on total bone marrow cells from 2 wild-type control mice (lanes 1-2), 2 preleukemic transgenic mice (lanes 3-4), and leukemic bone marrow cells from 2 leukemic transgenic SALL4B mice (lanes 5-6). Both c-Myc and Cyclin D1 expression were significantly up-regulated in SALL4B transgenic mice at both preleukemia MDS and leukemic stages. Beta actin was used as an internal standard.