FIG. 4.

P-TEFb-dependent induction of HEXIM1 expression by HMBA. (A) HMBA treatment of HeLa cells increases HEXIM1 expression. HEXIM1 levels in NE of HMBA-treated cells were detected by Western blotting at various time points of the treatment. The HEXIM1 signals were quantified and normalized to those of CDK9 for each time point, and they are shown as percentages relative to the pretreatment level, which was set to 100%. (B) P-TEFb is required for HMBA-induced HEXIM1 mRNA synthesis. HeLa cells transfected with either the empty pSuper vector (−) or vectors expressing the CDK9- and CycT1-specific siRNAs (siCDK9 and siCycT1, respectively) were treated with HMBA for 0, 6, or 24 h. Total RNA was isolated and subjected to Northern blot analysis to detect the major 2.5-kb and minor 4.0-kb HEXIM1 mRNA. The 18S and 28S rRNA present in total RNA were stained with ethidium bromide and used as loading controls. (C) CycT1- and Brd4-dependent production of the HEXIM1 protein. Western blotting was performed to examine the levels of Brd4, CycT1, HEXIM1, and CDK9 in NEs prepared from HeLa cells transfected with either the empty pSuper vector or the indicated siRNA-expressing pSuper constructs at 48 h posttransfection.