Figure 4. CDH11 expression is upregulated by endothelial cells and TGFβ.

(A) Primary human GBM cells are labeled with GFP and co-cultured with either unlabeled GBM cells or endothelial cells. GFP expressing cells are then purified by fluorescence activated cell sorting and CDH11 expression is measured by qRT-PCR. (B) Coculture with endothelial cells (HUVEC, mBend) causes increased expression of CDH11 in primary human GBM cells (GBM line 83 P = 0.0164 by ANOVA (n = 5); Line 77, P = 0.0284 by ANOVA (n = 6)). (C) CDH11 mRNA is increased in a dose-dependent fashion by TGFβ. (N = 3 for each cell line. Line 18 P<0.0001; Line 69A P = 0.0001; Line 71A P = 0.0005; all by Repeated Measures ANOVA). (D) Endothelial cell co-culture induces p-SMAD3 expression in GBM cells (top row: GBM co-cultured upon GBM; bottom row: GBM cultured with endothelial cells). Bar = 50 µm. (E) Western blots confirm induction of SMAD3 phosphorylation by co-culture of GBM with endothelial cells. (F) TGFβ transcriptional targets SMAD7 (Line 83, P = 0.0275 by ANOVA (n = 4); Line 77 n = 4) and Serpine-1 (Line 77, P = 0.0082 by Repeated Measures ANOVA (n = 3); Line 83 n = 4) are upregulated in GBM co-cultured with endothelial cells (primary HUVEC or mouse brain endothelial cell line mBend). (G) CD44 mRNA (Line 83 P = 0.0196 by ANOVA (n = 5); line 77 P = 0.0017 by ANOVA (n = 4)) and (H) cell surface expression (measured by flow cytometry) is upregulated after endothelial co-culture. For all pairwise comparisons, Newman Keuls posthoc tests were used, * P<0.05, ** P<0.01, *** P<0.001 and refer to comparison with control (GBM-GBM) unless otherwise noted.