Calaluce et al. BMC Cancer 2010, 10:126 http://www.biomedcentral.com/1471-2407/10/126 Page 7 of 15 Figure 3 GO Classification of genes found by RIP CHIP of potential HuR targets and their relationship to the Acquired Capabilities of Cancer Model. A. Differentially expressed genes which are more represented in the Biological Processes (BP) GO category than expected. B. Original representation showing subsets of transcripts found to be targets of association with HuR (normal type). New transcripts found in this study with RIP-Chip (bold type). Enhanced expression upon binding to HuR influences several of the acquired capabilities of cancer cells described by Hanahan and Weinberg [23,24]. For the complete GO analyses see Additional File 2, Table S1. In Table S1 we list the top HuR associated mRNAs in the different categories which were approxi- mately 5 fold enriched or greater. As can be seen in Fig- ure 3B, a partial listing of some of these genes (in bold) are candidate members to multiple areas of cancer con- trol, as suggested by Hanahan and Weinberg. We note that though b-ACTIN mRNA was amongst the most abundant of HuR-associated mRNAs in MCF-7 cells, b- ACTIN mRNA levels were only 3.93-fold higher in HuR IP compared to IgG IP in MB-231 cells. Therefore, since this was less than the 5-fold cut-off we employed for Table S1, it is not listed. Thus, these results may have identified novel HuR-controlled genes which may play roles in breast carcinogenesis in a cancer subtype- specific fashion. Validation of HuR targets CD9 and CALM2 by real-time PCR and biotin pull-down analyses In order to validate HuR binding to genes identified in Fig- ure 2, we chose two known cancer associated genes, CD9 and CALM2, highly expressed in both cell lines. Two inde- pendent approaches confirmed the physical interaction between HuR, CD9 and CALM2 mRNAs. Precipitated mRNA from the RIP-Chip experiments were analyzed by quantitative RT-PCR. Both CD9 and CALM2 mRNAs were enriched in the HuR RIP by as much as 160 fold (Figures 4A and 4B), but not the isotype control IP. We