Fig. 6. Endothelial cell proliferation is suppressed in Man1^GT/GT cells. (A,B) Immunohistochemistry of EBs, grown in suspension for 10 days then explanted for 6 days, using an antibody to PECAM-1. (A) Endothelial cells form extensive branches in the wild-type (+/+). (B) The Man1^GT/GT explants form fewer and less organized branches. (C) FACS analysis of PECAM-1⁺ cells isolated from Man1^GT/GT and wild-type EB explants, showing that in cultures of both genotypes the percentage of PECAM-low expressing cells was similar, but the percentage of PECAM-high expressing cells was strongly reduced in the Man1^GT/GT cultures. (D) TGFß1 further inhibits cell proliferation in Man1^GT/GT fibroblasts. Wild-type and Man1^GT/GT MEFs and SVEC cells were treated with TGFß1 for 72 hours at the indicated concentrations. Mean±s.e.m. (n=6 wells per treatment). Asterisks indicate statistically significant decrease in the proliferation of Man1^GT/GT MEFs compared with wild-type and SVEC cells. (E) Real-time gene expression analysis of EB explants. EBs were grown in suspension for 14 days then explanted for 4 days. RNA for each data point was derived from >20 EBs and assayed in triplicate. Data are shown as a ratio of Man1^GT/GT relative to wild type and were normalized to an average of S18 and Gapdh. Mean±s.e.m.; ^*, P<0.05; ^**, P<0.005; Student's t-test. Scale bar: 100 µm.