Figure 4

Functional validation of genes identified as potential modulators of aromatase inhibitor (AI) response when amplified and overexpressed. (A) Western blotting of lysates from SUM44 cells: each of the three genes, silenced and blotted for, was used to demonstrate siRNA efficacy and antibody specificity. (B) Panel of estrogen receptor (ER)-positive cell lines were used to assess the effect of RNA-interference-induced silencing of CHKA, LRP5 and SAPS3 on cell viability. Cell line names in red font harbor amplification of these genes; those named in black do not. Data for each knockdown (performed using SMARTpools) were normalized to readings from cells transfected with a non-targeting control and grown in dextran charcoal-stripped (DCC) media. Data are representative of six replicates from at least two independent experiments. Numbers indicate t-test P-value. (C) The same panel was used to assess the effect of RNA-interference-induced silencing of CHKA, LRP5 and SAPS3 on ER-driven proliferation. After growing and transfecting cell lines in DCC, cell viability was assessed as a surrogate marker of proliferation in the presence of increasing concentrations of estrogen (E2). Cell line names in red font harbor amplification of these genes; those in black do not. Data for each knockdown (performed using SMARTpools) were normalized to readings from cells transfected with a non-targeting control and grown in DCC media. Drug curves were inferred from non-linear regression. Error bars represent standard error of the mean. Data are representative of six replicates from at least two independent experiments. P-value is for one-way analysis of variance.