Fig. 3
Rasip1 knockdown (KD) in CD45lowc-Kithigh cells. A Upper panel: A schema of the position of the Rasip1 shRNA in the Rasip1 mRNA, a retrovirus vector of Sox17-IRES-mCherry, and retrovirus vectors of shLuc and shRasip1. Lower panel: After 4 days of Sox17-IRES-mCherry transduction, Sox17-transduced cells formed cell clusters. shLuc and shRasip1 were introduced into Sox17-transduced cells, and the cells were co-cultured with OP9 stromal cells. Morphologies of shRasip1 (green, GFP+)-transduced Sox17+ cells (red, mCherry+). Scale bar, 1.0 mm. B shLuc- or shRasip1-transduced GFP+ cells were recovered by FACS. Expression of Rasip1 in shLuc- and shRasip1-transduced cells was analyzed by RT-PCR and normalized by β-actin gene expression. RT-PCR result is in the static image that is generated by a mirror-reversal of an original across a horizontal axis, due to the sample order in the electrophoresis. C Sorted GFP+ cells (2.5 × 10.3) were embedded in a semisolid medium. The number of total (CFU-C) and mix (CFU-Mix) colonies was scored after 7 days of culture. The pair of colony numbers (sh-Luc and shRasip1) in each experimental group were plotted and connected by a line. Each symbol represents the colony numbers observed with each cell preparation from individual litters of E10.5 mice (n = 7, 7 individual litters of mice from which each experimental group was set up). *p ≤ 0.05; **p ≤ 0.01