Calaluce et al. BMC Cancer 2010, 10:126 http://www.biomedcentral.com/1471-2407/10/126 Page 11 of 15 Figure 7 Effects of over-expressing or reducing HuR on CD9 and CALM2 expression in MCF-7 cells. A. Western analysis of HuR over- expression in heterogenous population of cells reveals approximately 10% over-expression. B. Lentiviral HuR shRNA efficiently knocks down HuR protein by over 90%. C. HuR over- expression and under-expression results in small changes in CD9 protein levels in MCF-7 cells. D. Levels of both CD9 and CALM2 mRNAs are unchanged in cells which over-express HuR; whereas lentiviral knock-down of HuR in MCF-7 cells results in decreases in steady-state mRNA levels (E). The graph in (F) shows minimal changes in CD9 mRNA and protein levels in HuR over-expressing MCF-7 cells. The CD9 mRNA levels, however, are more affected in HuR knock-down. P value is < 0.005; N.S. = not statistically significant; * = statistically significant. Discussion We utilized RIP-Chip technologies to define differentially regulated HuR genes in ER+ and ER- breast cancer. To our knowledge, this is the first report of a side-by-side genome-wide comparison of HuR-associated targets in wild type ER+ and ER- breast cancer cells. Our findings indicated that HuR interacts with small subsets of genes in breast cancer, out of the possible 8% of human genes possessing AREs which are potential HuR targets. Three broad categories of HuR targets were identified. First, there was a subset of targets only found in ER+ breast cancer. Second, there was a unique subset of HuR targets found only in ER- breast cancer. A third subset consisted of HuR-associated mRNAs common to both forms of breast cancer, many of which were previously described as having roles in cancer. We selected and validated two HuR targets, CD9 and CALM2 mRNAs, which were found in high abundance in both types of breast cancer. Initially, we employed the pre- viously developed “heat map” signature of HuR binding to