Reduction in SP1 expression in U-87 MG cells (in cord assay) reduced tumor-driven cord formation.

<p>(A-B) U-87 MG cells were plated in permeable transwells and transduced with non-targeting shRNA (control) or pooled shRNA directed against SP1 for 72 hours prior to (A) whole cell protein extract isolation and Western blot analysis using antiserum directed against SP1 (61.3±9.0% knockdown) and β-actin as a loading control, and (B) movement of the U-87 MG cells into ADSC/ECFC co-culture receiver plates. Cord formation was assessed following 72 hours by immunohistochemistry for CD31 (green), α-smooth muscle actin (red), and Hoechst to stain nuclei (blue). Representative images (5× magnification) are shown. (C) U-87 MG cell viability assesment by cellular ATP production using Cell Titer Glo Assay. (D) ELISA analysis for VEGF secretion in U-87 MG conditioned media. Graphs represent mean ± standard error from three independent experiments, and asterisks denote statistically significant differences (*, <i>p</i><0.05; **, p<0.01; ***, p<0.001) compared to the non-targeting shRNA control vector.