ROSA26-targeted Flt-1 isoform transgenes partially rescue the endothelial cell mitotic index. (A–F) Day 7 differentiated cultures were fixed and stained for PECAM-1 (green), the mitotic marker antiphosphohistone H3 (red), and the nuclear dye DRAQ 5 (blue). (G) Representative confocal images were scored by counting the PECAM-positive nuclei and the percentage that reacted with the phosphohistone antibody to calculate the endothelial mitotic index. Error bars represent SEM. χ² analysis showed that the WT endothelial mitotic index was significantly different from flt-1^−/− mutants (χ² = 15.1; P ≤ 0.0001), and each of the Flt-1 isoform rescue clones differed from flt-1^−/− mutants (sFltR1: χ² = 12.2; P ≤ 0.0005; sFltR18: χ² =7.6; P ≤ 0.005; mFltR23: χ² = 7.5; P ≤ 0.008; mFltR25: χ² = 13.0; P ≤ 0.0003). Bar, 50 μm.