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Fig. 4 | BMC Cancer

Fig. 4

From: Increased Wnt5a in squamous cell lung carcinoma inhibits endothelial cell motility

Fig. 4

The effect of Wnt5a on VEGF-A induced endothelial cell activation and motility. a, CD105 mRNA expression is significantly higher in primary AC compared to SCC samples. Error bars, SEM. One-way ANOVA, post hoc Bonferroni; n = 11 and n = 12 per groups. b, Flow cytometric analysis of CD105 protein expression in CD31 positive endothelial cells in primary AC and SCC samples. n = 6 per groups. c, Flow cytometric analysis of CD105 levels in normal and high VEGF-A microenvironment in 3D lung aggregate tissues has also shown an increase of activation marker CD105 in VEGF-Ahigh tissues. The double positive (CD105/CD31) cell population was considered as activated endothelial cells. Independent samples t-test, n = 6. 1 μg/ml rhWnt5a treatment had no effect on the VEGF-A induced endothelial cell activation measured by the double positive (CD105/CD31) cell population identified by flow cytometric analysis. Independent samples t-test, n = 6. d, Localization of endothelial cells was identified by immunoflurescent staining of CD31 and analyzed by confocal microscopy in 3D lung tissue aggregates. In VEGF-Anormal microenvironment endothelial cells remained diffuse in the tissue. Under VEGF-A excess endothelial cell migrated towards the source (VEGF-Ahigh fibroblasts) of the signal in the center of the aggregate tissue. 1 μg/ml rhWnt5a treatment of VEGF-Ahigh tissue aggregates inhibited endothelial cell accumulation in the center of the aggregate. Bar chart represents the quantification of endothelial cell distribution. Relative area of CD31+ endothelial cells are compared to total field. Percentages were calculated as relative area of endothelial cells/area of total field *100. Error bars; SEM. Independent samples t-test n = 3. Representative images of three independent experiments are shown. Scale bars, 50 μm. e, HMVEC-L transwell migration assay. Endothelial cells migrate significantly faster towards VEGF-Ahigh fibroblast, while 1 μg/ml rhWnt5a can reverse the effect of elevated VEGF-A level. Scale bar 100 μm. One-way ANOVA, n = 3. P < 0.05 was considered as significant, * p < 0.05, ** p < 0.01, *** p < 0.001

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