BCR/ABL- and IL-3–induced VEGF gene expression in Ton.B210-X cells.

(A) Northern blot analysis. Starved Ton.B210-X cells were maintained in the presence (BCR/ABL) or absence (starved) of doxycycline and then subjected to Northern blotting. In addition, Ton.B210-X cells cultured in IL-3 were examined. Northern blot analysis was performed using a murine VEGF probe. After stripping the blot, a β-actin probe was applied. 28S indicates the position of 28S rRNA. (B) VEGF promoter activity. Ton.B210-X cells were transfected with the VEGF-Luc and pCMV-βGal plasmids and were grown in serum-free medium in the presence (BCR/ABL) or absence (starved) of doxycycline for 48 hours. In addition, cells were maintained in IL-3. After incubation, cells were harvested and assayed for the expression of luciferase and βGal activities. Luciferase activity was reported as the ratio VEGF-Luc/pCMV-βGal and was expressed as a percentage of control (starved cells). (C) VEGF ELISA. Supernatants of Ton.B210-X cells incubated with (BCR/ABL) or without (starved) doxycycline or cultured in IL-3 for 24 hours were collected and examined for the levels of secreted VEGF by ELISA. Results represent the means ± SD of 3 independent experiments.