Knockdown of Ape1 expression results in a decreased frequency of BL-CFC formation. (A) The distinct morphology between BL-CFCs and secondary EBs is depicted (× 20). (B) The individual BL-CFC or secondary EB colonies were collected separately. mRNA was extracted and RT-PCR assay revealed the distinct phenotype between these 2 cell types; BL-CFCs express Flk-1 and scl genes, but not the brachyury gene. In contrast, secondary EB cells express the brachyury gene, but not Flk-1 and scl genes. (C) Hemangioblast progenitor assay indicated that knockdown of Ape1 gene expression with Ape1 siRNA, but not scrambled siRNA day-3 EB cells, resulted in a decrease in BL-CFC formation. *P < .05 comparing Ape1 siRNA to scrambled siRNA, treatment with transfection reagent (OFA2000), or no treatment. Data represent the mean ± SD for 3 experiments. (D) Knockdown of Ape1 gene expression of day-3 EB cells resulted in an increase in secondary EB formation in the culture. *P < .05 comparing Ape1 siRNA to scrambled siRNA, treatment with transfection reagent (OFA2000), or no treatment. Data represent the mean ± SD for 3 experiments. The image in panel A was obtained using a Leica MZ9.5 microscope equipped with a Planapo 6× objective and a 10×/2.1 B aperature (Leica, Allendale, NJ). Image was captured using a Leica DFC320 camera with Leica Application software version 2.4.0.