Inhibition of Erg expression in endothelial cells. HUVECs (105) grown on gelatin-coated 6-well plates were transfected with either control GB or Erg-specific GB (100 nM) for 48 hours. (A) Erg mRNA levels from RNA extracts were quantified using RT-PCR, normalized to GAPDH. Erg mRNA expression was significantly reduced after GB treatment, ***P < .001. Values are means plus or minus SEM, n = 4. (B) Whole-cell protein extracts were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) immunoblotting with antibodies to Erg and tubulin. Erg protein expression was significantly reduced in the Erg-GB–treated cells, **P < .01. Values are means plus or minus SEM, n = 3. (C) Immunofluorescence analysis of the distribution and expression of Erg before and after GeneBloc treatment. Cells grown on gelatin-coated glass coverslips were transfected with GB as above. After 48 hours, cells were labeled and visualized for Erg (top) or the nuclear marker TOPRO-3 (bottom). Scale bar, 100 μm. (D) ICAM-2 mRNA levels, measured in RNA extracts from GB-treated HUVECs using RT-PCR, normalized against GAPDH, were significantly decreased after Erg inhibition, **P < .01. Values are means plus or minus SEM, n = 3. (E) ICAM-2 protein expression was also down-regulated in Erg-GB–treated HUVECs, as determined by SDS-PAGE immunoblotting using antibodies to ICAM-2, normalized against tubulin, *P < .05. Values are means plus or minus SEM, n = 3.
