ER71 inhibits canonical Wnt signaling. (A) iEr71 ES cells were differentiated in the absence (Control) or presence (+CHIR99021, 1μM) of CHIR99021 from days 2-3 of differentiation, either with (+DOX) or without (−DOX) DOX from days 2-3 of differentiation. Flk-1 and PDGFRα expression were analyzed at day 3. (B) Er71^−/− ES cells were differentiated and treated with CHIR99021 or DKK1 from day 2. Flk-1 and PDGFRα expression was analyzed at day 3. (C-D) ST2 cells were cotransfected with Lef1-luciferase ± MSCV-Er71 and ± Wnt3a conditioned medium (C) or ± β-catenin expression vector (D). Luciferase activity was measured 48 hours later. Firefly luciferase activity was normalized by Renilla luciferase activity. (E) Flk-1⁺ mesoderm sorted from iEr71 EBs was cotransfected with Lef1-luciferase and β-catenin. DOX was added to culture and luciferase activity was measured 48 hours later. (F) Flk-1⁺ mesoderm sorted from Er71^+/+ and Er71^−/− EBs were cotransfected with Lef1-luciferase ± MSCV-Er71. Luciferase activity was measured 48 hours later. Data are presented as the means ± SD of at least 4 independent experiments. *P < .05; **P < .01; *** P < .001.