RelB controls glioma cell motility and invasion.

<p><i>In vitro</i> scratch assays were performed to compare the motility of <i>(A)</i> U87 cells expressing shRNA control, shRelB-1 cells, shRelB-1+vector and shRelB+mRelB; <i>(B)</i> U87 cells expressing pLenti6 vector, pLenti6-mRelB, or pLenti6-hRelB. Photographs were taken of cells pre-scratch, 0 hours and 20–24 hours post-scratch. <i>(C)</i> Western blot was performed on U87 wild type or shRelB-3 cells using antibodies to RelB and actin. (D<i>)</i> Representative photographs of a side view of U87 cells invading three-dimensional collagen matrices. Arrow indicates the surface of the collagen matrix. <i>(E)</i> Average numbers of invading cells per field from 3 independent fields (+/− SD). <i>(F)</i> Average invasion distances (n = 100 cells) +/− SEM. <i>(G)</i> Representative photographs of a side view of U87-shRelB cell invasion +/− rescue with mRelB. <i>(H)</i> Quantification of number of invading cells from <i>G</i>. <i>(I)</i> invasion distance from G. Data shown are average numbers of invading cells per field from 3 independent fields (+/− SD). <i>(J)</i> Representative photographs of a side view of U87 cells overexpressing hRelB invading collagen matrices. <i>(K)</i> Quantification of invasion from J. Data shown are average numbers of invading cells per field from 3 independent fields (+/− SD). <i>(L)</i> Invasion distance from panel J.</p

RelB promotes glioma cell proliferation and survival.

<p><i>(A)</i> Western blot analysis of glioma cells using indicated antibodies. <i>(B)</i> Western blot analysis was used to assess RelB expression in U87 cells stably expressing a scrambled shRNA control or RelB targeting shRNAs. <i>(C)</i> MTS assays performed on U87 shRNA control, shRelB-1 and shRelB-3 cell lines. Error bars indicate standard deviation (SD), n = 4. <i>(D)</i> A Bioluminescent assay to measure Caspase 3/7 activity was performed on U87 cells expressing the indicated shRNA constructs. Error bars indicate SD. <i>(E)</i> Quantitative real-time PCR examining levelsof Bcl-2 and c-FLIP mRNA in RelB knockdown cells. Error bars indicate standard error (n = 3).</p

Kaplan-Meier curves from TCGA (The Cancer Genome Atlas) data analysis show the effect of RelB overexpression on time for tumor progression <i>(A)</i> and patient survival <i>(B)</i>.

<p>Kaplan-Meier curves from TCGA (The Cancer Genome Atlas) data analysis show the effect of RelB overexpression on time for tumor progression <i>(A)</i> and patient survival <i>(B)</i>.</p

RelB controls tumorigenesis glioma tumorigenesis <i>in vivo</i> and is a prognostic indicator of glioma patient survival.

<p><i>(A)</i> Subcutaneous xenografts of DiD-labeled U87 shControl and shRelB-1 cells were allowed to grow for 4 weeks (n = 4). Average volume of tumors was determined based on caliper measurement of tumor diameter. Inset shows representative <i>in vivo</i> tumor images taken with an <i>In Vivo</i> Kodak FX Imager. <i>(B)</i> Orthotopic intracranial injection of DiD-labeled U87 shControl, shRelB-1 and shRelB-3 cells were allowed to grow for 4 weeks. Representative <i>in vivo</i> tumor images from one experiment are shown (n = 3). <i>(C)</i> Western blot analysis was performed on patient-derived glioma cells. <i>(D)</i> Comparison of RelB protein and mRNA levels among the indicated cells. To compare RelB protein expression in U87 and BT cells, western blot data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057489#pone-0057489-g001" target="_blank">Figs. 1A</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057489#pone-0057489-g005" target="_blank">5B</a> were quantified and normalized to Actin. RelB mRNA levels were quantified by real-time PCR. <i>(E)</i> Intracranial tumor growth of DiD-labeled BT25 glioma cells expressing shRNA control or shRNA-RelB-3 was evaluated by <i>in vivo</i> fluorescence imaging 4 weeks after intracranial innoculation. Representative tumor images from one experiment are shown (n = 3). Similar results were seen with shRelB-1 cells (data not shown). (F) H&E and KI67 staining of frozen brain sections after 4 weeks of tumor growth. Yellow arrows indicate tumor borders. <i>(G)</i> Western blot analysis of RelB levels in BT25 shControl and shRelB-3 cells.</p