Figure 5

siRNA mediated downregulation of YB-1 reduced viral replication in infected CSC. (A) Immunblot of YB-1 in R28 cells 48 h after transfection with either control- or YB-1 specific siRNA. Tubulin serves as a loading control. (B) Quantification of YB-1 expression in R28 knockdown cells (normalized to tubulin). (C) R28 cells were transfected with control siRNA (siCo) or siRNA against YB-1 (siYB-1), respectively. 48 h later, cells were infected with 50 MOI of either Ad-WT or Ad-Delo3-RGD. Cells were harvested and DNA was isolated 4 h or 72 h after infection. Adenoviral DNA copy numbers as a degree of viral replication were assessed using real-time PCR. siRNA mediated knockdown of YB-1 by reduced Ad-WT replication in infected R28 cells to 76% and Ad-Delo3-RGD-replication to 42%. (D) Ad-Delo3-RGD does not replicate efficiently in human immortalized astrocytic SV-GA cells. Data represent the final viral titer as pfu/ml. Viral titers were determined by hexon titer assay from lysates of infected U87MG and SV-GA cells. 175.000 cells were infected with 10 MOI of Ad-WT or Ad-Delo3-RGD under serum-starved conditions (0.5% fetal bovine serum, no growth supplements) for 20 min. In the case of equal particle formation in both cell lines the ratio is = 1. Thus, a ratio >1 indicates higher viral production in astrocytes compared to U87MG cells. A ratio < 1 indicates lower viral production in astrocytes compared to U87MG cells (n=3, ** p=0.0028,)