Suppression of uPA and uPAR Attenuates Angiogenin Mediated Angiogenesis in Endothelial and Glioblastoma Cell Lines (original) (raw)
Figure 5
shRNA against uPA and uPAR inhibits nuclear localization of ANG and 45S rRNA gene expression and decreases ribonucleolytic activity in HMEC cells.
(A) Serum-starved endothelial cells (30 to 40% confluence) left untreated, treated with exogenous ANG (250 ng/mL for 48 hrs), pretreated with neomycin (100 µM for 1 hr), or treated with rANG and then with neomycin were taken and nuclear extracts were prepared and western blotted for ANG. The purity of the nuclear extracts was tested by probing for Lamin B (middle panel) and tubulin (cytoplasmic marker) (bottom panel). (B) Localization of ANG in nuclei of endothelial cells. Serum-starved (30 to 40%) cells were left untreated or transfected with pU2 for 48 hrs and stained for ANG (green) after permeabilization. Arrows indicate angiogenin. (C) puPA, puPAR and pU2 inhibit the ribonucleolytic activity of HMEC. Ribonucleolytic activity of ANG isolated from puPA-, puPAR- and pU2-transfected cells was done as described by Shapiro et al. *p<0.01, **p<0.001. (D) Fold induction of 45S rRNA gene expression in mock and empty vector-transfected cells was quantified using real-time RT-PCR. (E) HMEC cells (30 to 40% confluence) were left untreated, transfected with pEV, puPA, puPAR or pU2, and real-time RT-PCR was performed for 45S rRNA gene expression. The results shown in panel E represent the averages of standard deviation of data from three independent experiments that were normalized to the β actin expression levels. The 45S rRNA levels were normalized to uninfected cells and were assigned a value of “1” for comparison. *p<0.05, **p<0.001.