pp71 stimulates expression and secretion of the pro-angiogenic cytokine SCF.

<p><b>A</b>: NPC cultures were transduced with adenoviruses expressing either pp71 or GFP as a negative control and total RNA was harvested, reverse transcribed into cDNA, and analyzed using a quantitative PCR array. Fold-change in expression of 24 out of 87 cellular genes in pp71-transduced cells is displayed <b>B</b>: Conditioned medium from serum-starved NPCs, U87 cells, or normal human astrocytes treated as indicated were analyzed by ELISA for human SCF. *p<0.05 by student t-test when compared to untreated control; when compared to rAD-GFP control p = 0.037 for NHAs, 0.09 for NPCs, and 0.094 for U87s. <b>C</b>: Top panel: NPCs mock-infected or infected with HCMV TR strain for 5 days were immunostained for pp71 (red), SCF (green) and counterstained with DAPI (blue). Bottom panel: NPCs mock-infected or transduced with rAD-GFP or rAD-pp71 were immunostained for the pp71 associated HA tag (green – mock and rAD-pp71 only), human SCF (blue) and counterstained propidium iodide (red). <b>D</b>: Several cDNA samples as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068176#pone-0068176-g001" target="_blank">figure 1A</a> were analyzed by TaqMan for SCF expression. Copy number was determined using a human brain cDNA standard curve and is plotted relative to the presence of pp71 transcripts in the same specimens. <b>E</b>: Primary GBM cells were immunostained for pp71 (green) and SCF (blue). Secondary antibody only was a negative control (IgG-green) and counterstain was performed with propidium iodide (red). <b>F</b>: Frozen tissue sections from primary GBMs were processed by immunostaining for pp71 only (green) or pp71 and SCF (red, red arrow). Areas of co-localization in the merged image are yellow (yellow arrow). Tissues were counterstained with DAPI. Controls for immunofluorescent staining of frozen tissue sections were performed with secondary antibody only (anti-mouse IgG 488 and anti-rabbit IgG 594). <b>G</b>: The Pearson coefficient for green (pp71) and red (SCF) pixels were calculated in both co-stained and single-stained samples from 7 cells and plotted (quantification was performed as described in reference ²¹). pp71 and SCF staining was highly correlated (r = 0.69) and significantly different from single stained controls (*, p = 0.001 for pp71 and 0.002 for SCF).