FIGURE 11.

Effect of CTCF overexpression on ANG and RNASE4 expression. A, overexpression of CTCF with V5-His tag or FLAG-HA tag in cells. The V5-His tag was fused to the C terminus of the CTCF, whereas the FLAG-HA was fused to the N terminus of the gene. The vectors were transfected to DU145 cells, and the cell lysates were analyzed for transgene expression by Western blot (IB) with antibodies specific to HA or His. B, qRT-PCR analyses of ANG and RNASE4 mRNA level in CTCF-overexpressing cells. The mRNA level was first normalized to that of β-actin and then to control cell lines. The relative values to control cell line were shown. C, luciferase reporter activity of the full-length Pr-U construct in CTCF-overexpressing cell lines. The luciferase reporter construct was co-transfected with CTCF expression vector, and the luciferase activity was measured 48 h post transfection. D, effect of ANG and RNASE4 shRNA on gene expression of the ANG and RNASE4 locus. DU145 cells were infected by lentivirus particles encoding shRNA specific to ANG (E7 and E4) and RNASE4 (M2 and D10) as indicated in the top panel. Stable cells lines were selected by 1 μg/ml puromycin for 7 days. mRNA levels of ANG and RNASE4 were determined by qRT-PCR. mRNA levels were normalized to β-actin in the same sample. Data shown in the bar graphs in all panels are the means ± S.D. of three independent experiments.