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Fig. 4. Formation of colonies in 3D extracellular matrix by mouse mammary epithelial cells overexpressing Cripto-1 and ductal elongation in mammary glands from transgenic mice overexpressing Cripto-1 are both reduced in proximity to a Netrin-1 source. (A) Colony formation of EpH4/Cr-1 cells was assessed in Matrigel containing disks that were preabsorbed with PBS or rmNetrin-1 (200 ng/ml). Colonies were quantified in areas situated proximal (P), medial (M) or distal (D) from the sources as illustrated on the microphotographs (5x magnification). (B) Detail shows the area of growth inhibition (arrows) proximal to the disk preabsorbed with rmNetrin-1 (100 ng/ml) surrounded by a ring of EpH4/Cr-1 cells. (C) Summary of the results obtained from quantification of the colonies formed by EpH4/Cr-1. The mean number of colonies of at least 500 µm in diameter ({theta}=500 µm) was significantly reduced only in the areas in proximity to the rmNetrin-1 source (*P<0.05 compared to levels in the control group). White bars represent the number of EpH4/Cr-1 colonies in Matrigel containing PBS source and black bars, the number of colonies formed in Matrigel containing rmNetrin-1. Values are the mean ± s.d. of four separate experiments. (D) Whole mount morphology (10x magnification) of cholesterol pellets (*) releasing 25 ng/day of rmNetrin-1 shows a significantly reduced ductal elongation in the mammary gland of MMTV-CR-1 transgenic mice compared to cholesterol-only control pellets. The Netrin-1-releasing pellets did not affect ductal elongation in mammary glands of FVB/N mice. L, lymph node (E) Histogram summarizing the results of ductal elongation in mammary glands containing Netrin-1-releasing pellets. Distance was measured from the center of the mammary gland lymph node to the tip of the farthest growing duct and values are the mean with error bars indicating the s.d. from experiments in FVB/N (n=4) and MMTV-CR-1 (n=5) mice. A significant difference in elongation distance was observed between the two groups (P=0.008).





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