Expression of proximal and distal Runx1 during ES/EB differentiation. (A) Schematic representation of differences in the N-terminal amino acid sequences between distal and proximal Runx1 isoforms. The N-terminally located Runt domain binds DNA and core binding factor-β subunit. (B) Semiquantitative reverse-transcription PCR analysis of expression of distal and proximal Runx1 during ES/EB differentiation. (C) Schematic representation of Runx1 WT allele and double KI allele. Gray and white boxes indicate positions of Runx1 coding and noncoding parts, respectively. Arrows mark positions of PCR primers used for specific detection of both isoforms. (D) Fluorescence-activated cell-sorting (FACS) analysis of Flk-1 and proximal-hCD4 expression during EB differentiation between days 2 and 3.5. (E) FACS analysis of CD41 and proximal-hCD4 expression during EB differentiation between days 3 and 6. (F) FACS analysis of CD41 and distal-GFP expression during EB differentiation between days 4 and 6. (G left) FACS analysis of proximal-hCD4 and distal-GFP expression in presort (P1) and sorted populations (P2-P4) from day 6 EBs. (Middle) Numbers of hematopoietic colonies generated in methylcellulose by populations P1 to P4 from day 6 EBs. Error bars indicate SD of the mean (n = 3). (Right) FACS analysis of CD45 and distal-GFP expression 7 days after replating in methylcellulose media. Numbers represent percentages of respective populations.