Runx1 gene expression in blast colonies and BL-CFC.

(A) X-gal staining of Runx1^+/+ andRunx1^+/Z blast colonies. Colonies were generated from day 3.5 EB-derived cells and were stained after 4 days of growth. Original magnification × 200. (B) X-gal staining of nonadherent and adherent cells derived from Runx1^+/+ andRunx1^+/Z blast colonies. Arrowheads indicate LacZ⁺ adherent cells. Original magnification × 400. (C) Upper portion of figure shows FACS profile of FDG-stained day 3.75Runx1^+/Z EBs. LacZ-negative (Neg) and -positive (Pos) fractions isolated by cell sorting are indicated. Sorting gates were set by comparing the staining of the +/Z cells to the wild-type (+/+) cells. Middle portion shows number of blast colonies generated by the different sorted fractions. Colonies were scored at day 4 of culture. Bars represent standard error of the mean number of colonies from at least 3 cultures. RT-PCR gene expression analysis of LacZ-positive and -negative fractions of day 3.75 EBs is depicted in the lower portion. PCR products for Runx1 andβ-actin were detected by hybridization with specific³²P-labeled probes. Products for Brachyury,Flk-1, Scl, Gata-1, and β H1 were visualized following ethidium bromide staining and are presented as a negative image. (D) Cell sorting of FDG-stained day 3Runx1^+/Z EBs. Secondary EBs (2°EBs), transitional colonies (Trans), and blast colonies (Blast) were scored 4 days following replating of the isolated fractions. Bars represent standard error of the mean number of colonies from at least 3 cultures.