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. 2007 Mar 26;176(7):1035–1047. doi: 10.1083/jcb.200607053

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Copyright © 2007, The Rockefeller University Press

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Figure 1. — Knock-out of PDK1 affects early vascular development in EBs and EB-derived EC migration. (A) Representative phase-contrast images of EBs derived from PDK1^+/+ and PDK1^−/− ES cells at day 0 and 3 of differentiation. (B) EBs PDK1^+/+ and PDK1^−/− at 3, 7, and 10 d of differentiation were fixed and analyzed by indirect immunofluorescence with rat α-CD31 antibody; antigen–antibody complexes were detected with Cy2-conjugated donkey α-rat IgG. (C and D) FACS analysis of cells derived from EB PDK1^+/+ and PDK1^−/− at different differentiation stage (3, 7, and 10 d) to evaluate percentage of CD31- and Flk1-positive cells, respectively; data were plotted as the mean ± the SD of the percentage of positive cells from three independent experiments. (E) ES cells PDK1^+/+ and PDK1^−/− were differentiated into EBs for 3 d; they were then disaggregated, and cells were used in a chemotaxis assay, in the presence of a gradient of fibronectin (20 μg/ml) in combination (black bars) or not (white bars) with 30 ng/ml VEGF-A. Migration index is calculated assigning a value of 1 to the number of PDK1^+/+ CD31-positive cells that migrated toward fibronectin; data were plotted as the mean ± the SD of five independent experiments. Statistical significance (*, P < 0.01) is shown for VEGF-A–stimulated EC-PDK^−/− compared with EC-PDK1^+/+. Images are representative of five independent experiments. Bars, 100 μm.