NANOG knockdown decreases angiogenesis in Matrigel plug assays. (A) Timeline of the Matrigel plug assay. (B-D) HUVECs were incubated with the indicated shRNAs (retrovirus) for 10 hours (in presence of serum and growth factors), after which time the cells (105) were mixed with growth facto–reduced Matrigel (250 μL) plus WNT3A (50 ng/mL) and injected subcutaneously into athymic nude mice (n = 5). After 7 days, the Matrigel plugs were collected, embedded in paraffin, sectioned (4-5 μm), and stained with hematoxylin and eosin. Pictures of Matrigel plugs were taken using a Canon Powershot A640 digital camera controlled by Zoom Browser Ex software 5.7. Digital images were then saved as EPS documents using Adobe Photoshop CS. Multiple images were assembled using QuarkXpress 8.0 software and labeled, and final images were saved as EPS documents. (E-G) Note the prominent microvessels in the control shRNA (noninterfering shRNA retrovirus) plugs and the reduced number of capillaries in the NANOG knockdown plugs. FLK1 cDNA expression partially compensated for the loss of NANOG. Scale bar, 40 μm. (H) Quantification of microvessel capillaries in the Matrigel plugs. The data represent the mean ± SEM; n = 5-10 from 3-5 independent experiments; *P < .05 and **P < .01 compared with control or as indicated. (I-K) Thin cryosections (5 μm) prepared from Matrigel plugs were stained with anti–human von Willebrand Factor (red) or anti–mouse CD31(green) to distinguish human and mouse ECs. Highly autofluorescent red blood cells inside neovessels are yellow in color. Note that close association between human and murine ECs are indicated by white arrows. Green arrows indicate HUVECs that failed to form blood vessels; yellow arrow indicates neovessel made of HUVECs. Original magnification, ×200. Epifluorescence images were captured using a Zeiss Axioplan 2 inverted microscope under 20× objectives, using an AxioCam H digital camera. Digital images were saved as TIFF documents using Adobe Photoshp CS. Multiple images were assembled using QuarkXpress 8.0 software and labeled, and final images were saved as EPS documents.
