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. 2010 Aug;177(2):1004–1016. doi: 10.2353/ajpath.2010.090703

Figure 5.

Figure 5

Expression of endothelin-1 (ET-1) is up-regulated in podoplanin-overexpressing MCF7 breast carcinoma xenografts and ET-1 promotes migration and proliferation of lymphatic endothelial cells expressing endothelin receptor B. A: Relative human ET-1 mRNA levels, measured by real-time RT-PCR in MCF7 tumors derived from podoplanin-overexpressing (PDPN) or control (CTRL) cell clones. Data represent mean ± SD (n = 3), with the value for clone CTRL 9 set to 1. B: Migration of primary human lymphatic endothelial cells in the presence of 1 or 10 nmol/L recombinant human ET-1 or 20 ng/ml human VEGF-A in a two-chamber migration assay for 4 hours at 37°C. Data represent mean fluorescence from calcein-stained transmigrated cells ± SEM (n = 3). **P = 0.001, *P = 0.025. C: Proliferation of lymphatic endothelial cells in the presence of different concentrations of ET-1 and/or VEGF-A for 48 hours. Data represent mean fluorescence from 4-methylumbelliferyl heptanoate-stained live cells ± SEM (n = 3). *P = 0.05. D: Relative endothelin receptor B (ENDRB) mRNA levels, measured by real-time RT-PCR in primary human lymphatic (LECs) and blood vascular endothelial cells (BECs). Data are represented as mean +/- SD (n = 3), with the value for BEC set to 1. E: Immunoblot detection of ENDRB, immunoprecipitated from lysates of primary human LECs and BECs (500 μg of total protein). β-actin served as control for equal loading.