Gli3 repressor mediates the effect of cyclopamine in hEB-derived hematopoiesis. (A) Western blot for Gli3 showing the full-length (Gli3F) and the Gli3 repressor (Gli3R) forms. In cyclopamine-treated hEBs, the majority of the protein was processed to the shorter Gli3R form. (B) Gli3F: Gli3R ratio demonstrating the processing of protein to the Gli3R form after cyclopamine treatment. (C) Immunostaining for Gli3 in frozen sections from day 5 hEBs showing that cyclopamine treatment induces focal nuclear localization of Gli3. Scale bars indicate 10 μm. (D) Down-regulation of the Gli3 transcript 2 days after Gli3 siRNA transfection. (E) Western blot for Gli3 showing a reduction in the Gli3F and the Gli3R forms at day 4 after transfection. (F) Reduction of mature blood cells (%CD45⁺) 15 days after transfection. (G) EBs treated with cyclopamine (left) or transfected at ES and EB stages with Gli3R expression vectors exhibit similar increases in CD45⁺ blood cells. (H) Cyclopamine-treated and Gli3R-overexpressing cells also have augmented expression of the transcription factors Sox6, Bcl11A, and Myb, which are associated with definitive erythropoiesis. Suppression of embryonic HBE globin transcription is also apparent. Bottom panels for A and E are anti-tubulin loading controls. *P < .05; ***P < .001.