Figure 6

Inhibition of PDGFR/Abl suppresses ER/ERE-mediated transactivation. Cells were transfected with an artificial luciferase reporter and treated as indicated. Luciferase activity was measured and normalized to DCC to quantify ER-transactivation. A and B show the effect of nilotinib on ER-mediated transcription in wt-MCF7. C and D show the effect of nilotinib on ER-mediated transcription in LTED cells. The data shown are representative of four individual experiments. Bars represent ± SEM of triplicate samples. E. ChIP analysis to determine ER, AIB1 and CBP recruitment to the GREB1 promoter was performed. Cell monolayers were serum starved for 24 hours and treated for 45 minutes with E2 (1 nM) and nilotinib (4 μM) as indicated. Antibodies against total ERα, AIB1 and CBP were used to pull-down protein complexes and to assess their recruitment to the ERE located in the GREB1 promoter by q-PCR. Data shown are representative of two independent experiments. Abl, Abelson tyrosine kinase; AIB1, amplified in breast cancer 1; CBP, CREB binding protein; ChIP, chromatin immunoprecipitation; DCC, dextran charcoal-stripped bovine serum; E2, estradiol; ER, estrogen receptor; ERE, estrogen response element; LTED, long term estrogen deprived; SEM, standard error of the mean; wt, wild type.