Figure 5
Figure 5. Role of S100A10 in plasmin-dependent Matrigel invasion. WT and S100A10−/− mice were implanted with a Matrigel plug containing 200 ng/mL bFGF and 60 U/mL heparin. CD31 staining (green) of endothelial cells shows decreased invasion into the matrigel plug in S100A10−/− mice (A). Nuclei were stained with DAPI (blue). Tissue surrounding the matrigel plug is visible in the S100A10−/− sections. Quantification of positive CD31 staining of 20× fields from 3 separate matrigel plugs was performed with ImageJ software (B). T241 fibrosarcoma cells were injected subcutaneously into WT and S100A10−/−. Tumors were collected after 3 weeks. CD31 staining (green) of endothelial cells shows decreased staining of endothelial cells in tumors collected from the S100A10−/− mice (C). Nuclei were stained with DAPI (blue). Quantification of positive CD31 staining of 20× fields from 3 separate tumors was performed with ImageJ software (D). Sections were mounted with Vectashield mounting medium (Vector Laboratories) and viewed using a 20×/0.5 NA objective lens. Images were captured by the Zeiss Axioplan 2 microscope with a Spot 2 digital camera. Digital acquisition of the images was performed with Axiovision 4.7 (Zeiss). Primary WT or S100A10−/− murine endothelial cells (E,G) or control or S100A10-depleted TIME cells (F,H) were added to the top chamber of Transwell chambers in the presence of media and in the presence or absence of Pg (0.5μM). Some chambers were coated with Matrigel (invasion assays; E,F) or uncoated (migration assays; G,H). The lower chambers contained media with 10% FBS. Cells were incubated for 48 hours, after which invading cells were stained with H&E and counted. Data are expressed as mean number of cells per 40× field ± SD of 3 independent experiments. Statistical analysis was performed by use of the Student t test (B,D,E,G) or ANOVA (F,H); ***P < .001. In some experiments cells were pretreated with CpB (5 U/mL), which was added to the upper chamber where indicated. Figures were generated with Adobe Photoshop CS3 Version 10 (Adobe Systems Incorporated).

Role of S100A10 in plasmin-dependent Matrigel invasion. WT and S100A10−/− mice were implanted with a Matrigel plug containing 200 ng/mL bFGF and 60 U/mL heparin. CD31 staining (green) of endothelial cells shows decreased invasion into the matrigel plug in S100A10−/− mice (A). Nuclei were stained with DAPI (blue). Tissue surrounding the matrigel plug is visible in the S100A10−/− sections. Quantification of positive CD31 staining of 20× fields from 3 separate matrigel plugs was performed with ImageJ software (B). T241 fibrosarcoma cells were injected subcutaneously into WT and S100A10−/−. Tumors were collected after 3 weeks. CD31 staining (green) of endothelial cells shows decreased staining of endothelial cells in tumors collected from the S100A10−/− mice (C). Nuclei were stained with DAPI (blue). Quantification of positive CD31 staining of 20× fields from 3 separate tumors was performed with ImageJ software (D). Sections were mounted with Vectashield mounting medium (Vector Laboratories) and viewed using a 20×/0.5 NA objective lens. Images were captured by the Zeiss Axioplan 2 microscope with a Spot 2 digital camera. Digital acquisition of the images was performed with Axiovision 4.7 (Zeiss). Primary WT or S100A10−/− murine endothelial cells (E,G) or control or S100A10-depleted TIME cells (F,H) were added to the top chamber of Transwell chambers in the presence of media and in the presence or absence of Pg (0.5μM). Some chambers were coated with Matrigel (invasion assays; E,F) or uncoated (migration assays; G,H). The lower chambers contained media with 10% FBS. Cells were incubated for 48 hours, after which invading cells were stained with H&E and counted. Data are expressed as mean number of cells per 40× field ± SD of 3 independent experiments. Statistical analysis was performed by use of the Student t test (B,D,E,G) or ANOVA (F,H); ***P < .001. In some experiments cells were pretreated with CpB (5 U/mL), which was added to the upper chamber where indicated. Figures were generated with Adobe Photoshop CS3 Version 10 (Adobe Systems Incorporated).

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