Fig. 2

Immunophenotypic conversion of Scl-null FLK1-single positive cells. a Days 3.5/4 ES cell-derived mesodermal populations are functionally defined by expression of cell surface markers FLK1 and PDGFRα. P-SP, PDGFRα single positive; DP, double positive; F-SP, FLK1 single positive; DN, double negative. b (i) Distribution of FLK1- and PDGFRα−positive populations in WT day 3.5 EBs. (ii) SCL protein expression in day 3.5 WT EBs (intra-cellular FACS). (iii) Left, top, distribution of FLK1/PDGFRα-positive cells shown in bi and gated on SCL-positive cells; bottom, mean of 9 independent experiments; Right, top, distribution of SCL⁺ cells in each FLK1/PDGFRα compartment. Blue events: SCL⁺ cells, red events: SCL^- cells; bottom, mean of 9 independent experiments. c Re-aggregation assays. F-SP populations were FACS-sorted from WT and Scl^-/- EBs (left panels, day 3.5; right panels, day 4.5), allowed to re-aggregate for 24 h and analysed for FLK1/PDGFRα expression. The arrows show the different immunophenotypic conversions of WT and Scl^-/- cells at day 3.5 + 24 h. Bottom, mean of two independent experiments. d Top, distribution of FLK1-positive and PDGFRα-positive populations in Scl^-/- day 3.5 EBs; bottom, comparison with WT cells (shown in bi), mean of 4 independent experiments. e Scl:mCherry and Scl^Δ/Δ:mCherry reporter lines analysed in day 4.5 EBs. Representative FACS plots of mCherry expression (left), FLK1/PDGFRα expression (middle) and FLK1/PDGFRα plots gated on mCherry-positive cells (right) are shown. Below, mean of 2 independent experiments. Mean ± SD is shown in b–e; student’s t-test, *p < 0.05, **p < 0.01. Scale bars, 100 μm. See also Supplementary Fig. 3