Fig. 4

Rasip1 knockdown (KD) in Sox17-transduced CD45^lowc-Kit^high cells. A Morphologies of GFP⁺ cell clusters. Yellow arrowheads indicate cell clusters which expressed in bright green color (GFP⁺ with high intensity). The bar graph shows the average volume and standard deviation (n = 3, 3 individual litters of mice from which each experimental group was set up). Scale bar, 200 µm. The average volume of cell clusters with high GFP expression in each group (shLuc and shRasip1) was measured and analyzed as follows: (1) we measured the longest diameter and perpendicular diameter with the longest diameter; (2) we calculated the average volume as follows: cluster volume = length × width²/2, where length represents the largest cluster diameter and width represents the perpendicular diameter with the longest diameter; (3) we calculated the average volumes of 10 clusters in maximum in one individual experiment; (4) we calculated the total average volume of all 3 experiments (n = 3, 3 individual litters of mice from which each experimental group was set up). B Upper panels: shLuc- or shRasip1-transduced cells with relatively high GFP⁺ expression levels, i.e., with the relatively high expression of the sh constructs were recovered by FACS. As indicated in the FACS profile, the dark green squares show the high GFP⁺ cell population. 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. Lower panels: Sorted GFP⁺ cells (3.0 × 10.³) were embedded in a semisolid medium. The number of total (CFU-C) and multilineage (CFU-Mix) colonies were 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 = 4, 4 individual litters of mice from which each experimental group was set up). *p ≤ 0.05