Effects of CDK9 inhibition on the phosphorylation of the CTD of RNAPII and the expression of genes in hTERT-immortalized normal human fibroblasts. CDK9 activity was inhibited in BJ-TERT fibroblasts via adenoviral mediated transduction of a tetracycline-repressible (tet) dominant negative CDK9 mutant (DN) or by pharmacological treatment with 300 nM flavopiridol (FVP). No functionally significant DN expression occurs in the presence of tetracycline (lanes 3 to 10). FVP treated cells were also previously transduced with the same adenoviruses and cultured in the presence of tetracycline (no DN effect) to normalize for viral effects as described in the text. All treatments were done in triplicate and duplicate samples are shown in A and B. The other replicate performed separately but under the same conditions exhibited virtually the same effects (not shown). Ectopic expression of dnCDK9 or treatment with FVP inhibit both RNAPII Ser-2 and Ser-5 phosphorylation. (A) and the expression of HEXIM1 transcripts. (B) as determined by western and northern blot analysis, respectively. Coomassie Blue (A) and EtBr (B) stains are shown for loading controls. (C) Global gene expression effects of ectopic expression of dnCDK9 or FVP treatment in BJ-TERT fibroblasts. Normalized Affymetrix microarray data (log2 ratios, see the text in the results section) for all transcripts of triplicate samples were analyzed by correlation uncentered, average linkage, hierarchical clustering (left heat map). Transcripts whose levels changed +/- 1 log2 in any treatment were reclustered (middle heat map) and the heat map was magnified for clarity (right heat map). See the text in the results section for details. Correlations are shown on the top of the arrays. A heat map legend is shown.