Figure 1.

P-TEFb and an intact SL region of intron I are required for the transcriptional regulation of MYB reporter constructs by estrogen. (A) HEK293 cells are transfected (∼0.5 × 10⁶/well of six-well plate) with 1.0 µg of WT-MYB CAT (Left panel) or 1.0 µg ΔSL-MYB CAT construct (Right panel) along with 0.1 or 0.2 µg each of CyclinT1 and CDK9 and 10 ng pCMV β-actin-promoter-luciferase expression plasmid. After 24 h, cells were incubated with fresh medium supplemented with or without 10 nM estradiol for 16 h. (B) Transient transfections in HEK293 cells were carried out, as described above, in the presence of either WT P-TEFb (0.1 µg each of CyclinT1 and CDK9) or dominant negative CDK9 expression construct (0.1 µg) along with WT P-TEFb complex before adding estrogen to the medium. (C) After transient transfection, cells were either exposed to estrogen or to 25 µM DRB as indicated for 16 h. In each case, CAT activity was normalized to β-actin-promoter-Luc activity as an internal control. Experiments were done in triplicate and repeated at least thrice.