Figure 1
Figure 1. High VEGF signaling leads to an increased frequency of excess centrosomes in endothelial cells of developing vessels. (A-B) Day 8 ES cell–derived WT and flt-1−/− mutant vessels were stained for PECAM-1 (red); note the vessel overgrowth and dysmorphogenesis in panel B. (C-J) Endothelial cells of WT and flt-1−/− mutant vessels were analyzed for centrosome numbers. (C-D) Day 8 ES cell cultures were fixed and stained for γ-tubulin (red), PECAM-1 (green), and DRAQ5 nuclear dye (blue). (F-G) Day 8 ES cell cultures that were WT or flt-1−/− and carried a PECAM-H2B::GFP transgene (green) were dissociated and attached to tissue culture dishes before fixation and staining for γ-tubulin (red). (E) Percentage of PECAM-H2B::GFP-positive cells with more than 2 centrosomes (WT, n = 159; flt-1−/−, n = 336). (I-J) WT and flt-1−/− embryos were harvested at E9.5, and yolk sacs were stained for γ-tubulin (red), PECAM-1 (green), and DRAQ5 nuclear dye (blue). (H) Percentage of PECAM-positive yolk sac endothelial cells with more than 2 centrosomes (WT, n = 88; flt-1−/−, n = 180). (L-M) HUVECs were incubated for 96 hours in low or high VEGF and then stained for γ-tubulin (red) and DRAQ5 nuclear dye (blue). (K) Percentage of HUVECs with more than 2 centrosomes (low VEGF, n = 2393; high VEGF, n = 3011). Arrows point to areas of cells with more than 2 centrosomes, and arrowheads point to areas of cells with 1 or 2 centrosomes. (D,I-J,M) Insets: Centrosomes at higher magnification. (A-B) Scale bar represents 50 μm. (C-M) Scale bar represents 5 μm. All experiments were performed at least 3 times. *P < .05, low VEGF vs high VEGF. ***P < .0001, low VEGF vs high VEGF. (A-B) Panels imaged with Olympus IX50 and 10×/0.25 NA CPlan RT objective; samples were in Aqua/Polymount (Polysciences); images acquired with Olympus DP71 camera and DP Controller Version 3.1.1.267 software. (C-M) Panels imaged with Zeiss LSM 5 Pascal and 63×/1.4 NA oil objective; samples were in Aqua/Polymount; images acquired with PASCAL Release Version 4.2 SP1 software. All images managed in Abobe Photoshop CS 2 9.0.

High VEGF signaling leads to an increased frequency of excess centrosomes in endothelial cells of developing vessels. (A-B) Day 8 ES cell–derived WT and flt-1−/− mutant vessels were stained for PECAM-1 (red); note the vessel overgrowth and dysmorphogenesis in panel B. (C-J) Endothelial cells of WT and flt-1−/− mutant vessels were analyzed for centrosome numbers. (C-D) Day 8 ES cell cultures were fixed and stained for γ-tubulin (red), PECAM-1 (green), and DRAQ5 nuclear dye (blue). (F-G) Day 8 ES cell cultures that were WT or flt-1−/− and carried a PECAM-H2B::GFP transgene (green) were dissociated and attached to tissue culture dishes before fixation and staining for γ-tubulin (red). (E) Percentage of PECAM-H2B::GFP-positive cells with more than 2 centrosomes (WT, n = 159; flt-1−/−, n = 336). (I-J) WT and flt-1−/− embryos were harvested at E9.5, and yolk sacs were stained for γ-tubulin (red), PECAM-1 (green), and DRAQ5 nuclear dye (blue). (H) Percentage of PECAM-positive yolk sac endothelial cells with more than 2 centrosomes (WT, n = 88; flt-1−/−, n = 180). (L-M) HUVECs were incubated for 96 hours in low or high VEGF and then stained for γ-tubulin (red) and DRAQ5 nuclear dye (blue). (K) Percentage of HUVECs with more than 2 centrosomes (low VEGF, n = 2393; high VEGF, n = 3011). Arrows point to areas of cells with more than 2 centrosomes, and arrowheads point to areas of cells with 1 or 2 centrosomes. (D,I-J,M) Insets: Centrosomes at higher magnification. (A-B) Scale bar represents 50 μm. (C-M) Scale bar represents 5 μm. All experiments were performed at least 3 times. *P < .05, low VEGF vs high VEGF. ***P < .0001, low VEGF vs high VEGF. (A-B) Panels imaged with Olympus IX50 and 10×/0.25 NA CPlan RT objective; samples were in Aqua/Polymount (Polysciences); images acquired with Olympus DP71 camera and DP Controller Version 3.1.1.267 software. (C-M) Panels imaged with Zeiss LSM 5 Pascal and 63×/1.4 NA oil objective; samples were in Aqua/Polymount; images acquired with PASCAL Release Version 4.2 SP1 software. All images managed in Abobe Photoshop CS 2 9.0.

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