Figure 2.

Ishikawa cells were transiently transfected with ERα. After recovery overnight, the cells were stimulated with ethanolic vehicle [−E₂ (control)] or 100 nM E₂ for 15 h without actinomycin D (−Act D) before the first time point [0 h; −E₂ (□) and +E₂ (●)]. Act D (5 μg/ml) was then added to the remaining cell cultures, and total RNA was extracted after an additional 2, 4, 6, 8, and 12 h (●). Control samples (without Act D) were assessed after 12 h (○). RT-PCR was performed as described and visualized on 4% agarose gels stained with ethidium bromide. Data of three independent experiments (±SD) were analyzed as ratio of VEGF:glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and expressed as percentage of the control. VEGF mRNA half-life was calculated as 4 h.