FIGURE 4.

Inhibition of SP-1 DNA binding, introduction of a nonfunctional SP-1 protein (ΔSP-1) or an SP-1 shRNA construct will prevent VEGF secretion and transcription. A and B, NSCLC, VEGF secretion (A) and mRNA accumulation (B) are inhibited in by mithramycin A. NSCLC were treated with 1 × 10⁻⁶ m mithramycin A for 24 h, and the supernatants were assayed by ELISA for VEGF. Total RNA was extracted, and the levels of VEGF mRNA were assessed by RT-QPCR with reference to GAPDH as a fold over untreated control. Open bars, A549; gray bars, H460; lined bars, H1299; black bars, MORP. NSCLC were co-transfected with 0.1 μg of VEGF-2068-luciferase or VEGF-135-luciferase plasmids with 1.0 μg of SP-1 DNA-binding domain construct (ΔSP-1) for 24 h followed by a luciferase assay. C–F, A549 (C), H460 (D), H1299 (E), and MORP (F). NSCLC were co-transfected with 0.1 μg of VEGF-2068-luciferase (2068) with 0.25 or 0.5 μg of SP-1 shRNA construct (pURSP-1) for 48 h followed by a luciferase assay. G–J, A549 (G), H460 (H), H1299 (I), and MORP (J). All of the measurements represent the means ± S.E. of three independent experiments. RLU, relative light units.