Figure 3.
2 μM PHA-767491 inhibits CDK9. A, 2 μM PHA-767491 blocks serine-2 phosphorylation independently or in combination with mithramycin. Immunoblot showing RNAPII and RNAPII CTD phosphoserine-2 relative to GAPDH loading control in TC32, TC252, and TC71 cell lines following exposure to medium (M), solvent (S), 100 nM mithramycin (100), 20 nM mithramycin (20), 2 μM PHA-767491 (P), or a combination of 20 nM mithramycin and 2 μM PHA-767491 (C) for 18 hours. Data representative of three independent experiments. B, 2 μM PHA-767491 induces the expression of endogenous retroviral RNA (ERV). Data represents fold change in expression (2ΔΔCT) of ERV-F and ER9–1 relative to GAPDH in TC32 (n=3), TC252 (n=3), and TC71 (n=3) cells following exposure to medium (M), solvent (S), 100 nM mithramycin (100), 20 nM mithramycin (20), 2 μM PHA-767491 (P), or a combination of 20 nM mithramycin and 2 μM PHA-767491 (C) for 18 hours. C, Schematic of nuclear run on assay used to measure RNA processivity. Primer pairs to both a proximal and distal region on the EZH2 locus were used for RT-qPCR. D, Processivity of RNA as measured by qPCR enrichment of mRNA from the proximal (start) vs. distal (end) amplicon of EZH2 relative to solvent after a TC32 nuclear run-on assay. Nuclei were exposed to 2 μM PHA-767491 during the run-on reaction (PHA), 20 nM mithramycin during the run-on reaction (MMA Run on), or cells were pretreated with 20 nM mithramycin for 18 hours before the run-on reaction (MMA Pre). Each biological replicate in the figure had three technical replicates. * = P < 0.05, ** = P < 0.01, *** = P <0.001, **** = P < 0.0001, error bars show standard deviation.