Figure 4.

Effect of CDK9 inhibition on phosphorylated pol II occupancy, transcription and chromatin looping. (A) Western blot analysis of Ser2-P pol II and total pol II levels in vehicle-, DRB- or Flavopiridol (Flavo.)-treated MEL cells. Valosin Containing Protein (VCP) served as a loading control. (B) Myb primary transcripts measured by RT–qPCR after treatment with the indicated compounds. Signals were normalized to 18S rRNA expression and transcript levels in vehicle-treated cells were set to 100%. (C) ChIP analysis of Ser5-P and Ser2-P pol II binding at the Myb transcriptional unit in vehicle-, DRB- and Flavopiridol-treated MEL cells. Genomic coordinates, gene location, CTCF occupancy and PCR primers are indicated above each graph (as in Figure 3). (D) Time-course CDK9 inhibition in MEL cells using DRB and Flavopiridol. Myb 5′ end and 3′ end transcripts were measured by RT–qPCR. Primer locations within the gene are depicted by coloured rectangles in a schematic below the graphs. (E) 3C-qPCR analysis on vehicle- or DRB-treated MEL cells. The Myb promoter HindIII fragment was used as a viewpoint. Data are plotted as mean±s.e.m. of at least two independent experiments.