FIGURE 5.

NSCLC cells express higher levels of SP-1 total cell protein than normal human bronchial epithelial cells. Native SP-1 transcription factor binding to the VEGF promoter increases with VEGF expression as SP-3 transcription factor binding decreases. Increased SP-1 binding is paralleled by increased histone H4 acetylation and histone acetyltransferase binding to the VEGF promoter of NSCLC cells. A, 30 μg of total cellular protein from NHBE and A549 were Western blotted and probed with an anti-SP-1 antibody (left panel). 10 μg of total cellular protein from A549, H460, H1299, and MORP were Western blotted and probed with an anti-SP-1 antibody (right panel), and the blots were stripped and reprobed with an anti-GAPDH antibody. B and C, the images are representative blots from experiments repeated three times. ChIP was performed for transcription factors SP-1 (B) and SP-3 (C) from chromatin samples from NSCLC cells maintained in serum-free medium for 24 h. IP and input DNAs were subjected to QPCR for the VEGF promoter between −85 and + 50 bp relative to the transcription start site of the VEGF promoter. Control ChIP was performed with equivalent rabbit IgG for all chromatin samples. D, ChIP for identical samples and their relevant control IgGs (open bars) were performed for acetylated (black bars) and nonacetylated histone H4 (cross-hatched bars), acetylated (diagonal line bars), and nonacetylated histone H3 (horizontal line bars). E, p300 and histone acetyltransferase. F, P/CAF and histone acetyltransferase. G, CBP and histone acetyltransferase. All of the measurements represent the means ± S.E. of three independent experiments.