Figure 5

Mechanistic assessment of the effect of CHKA in modulating response to aromatase inhibitor (AI) therapy. (A) Following RNA-interference-mediated silencing of CHKA, SUM44 cells were transfected with an estrogen receptor (ER)/ERE luciferase reporter construct, and then treated with E2 or dextran charcoal-stripped media (DCC) for 2 days before reading luciferase activity. Data are normalized to the activity in the DCC-treated control transfected cell lines. *Significant P-value (<0.05) between the indicated column and corresponding siControl-equivalent. (B) To validate effects seen with the ER-ERE reporter assay, expression levels of two well known ER-regulated genes (TFF1 and GREB1) were assessed by quantitative real-time PCR following RNA-interference-induced silencing of CHKA. Data normalized to DCC-treated control transfected cell lines; *P-value <0.01 between indicated column and corresponding siCON equivalent. (C) Following RNA-interference-induced silencing of CHKA, cell lysates were subjected to gel electrophoresis and western blotting using indicated antibodies. Cells were treated with 1nM E2 for 1 h or 24 h following transfection, to represent the two phases of ER dynamics (early active- and late turnover phase). Blots are representative of at least two independent experiments; numbers below each band represent densitometry analysis of intensity, measured as a ratio of the siControl with no siCHKA or E2 treatments.