Calaluce et al. BMC Cancer 2010, 10:126 http://www.biomedcentral.com/1471-2407/10/126 Page 10 of 15 Figure 6 HuR differentially regulates CD9 and CALM2 in MB-231. A. Epitope HA tagged HuR is over-expressed by 142% and 138% respectively, in stably transfected clones 4E1 and 5F1, as compared to empty vector (EV) control clone 2C7. B. HuR knock-down using lentiviral short hairpin (sh) RNA H760 results in a 94% reduction in steady state levels of protein in clone A7 (LL = lentilox control). C. HuR over-expression results in a 40% reduction in CD9 protein levels as assayed by Western analysis; however, HuR knock-down using lentiviral shRNA results in an increase from 100% to 228% of CD9 levels. D. Over-expression of HuR decreases CD9 mRNA levels but not CALM2 expression. Analysis of steady state CD9 and CALM2 mRNA levels by quantitative RT-PCR reveals significant decreases in CD9 mRNA levels, whereas CALM2 levels are unaffected. Although CALM2 expression appears greater, the change is not significant. E. Knocking down HuR levels by shRNA in MB-231 cells shows significant increases in CD9 and CALM2 mRNA levels by quantitative RT-PCR. Decreased levels of HuR mRNA validate HuR shRNA knock- down. F. Graph showing the effects of HuR on the expression of CD9 mRNA. HuR over-expression results in decreases in both mRNA and protein levels, though the decreases are greater in RNA. Whereas, HuR knock-down by shRNA results in significant increases at both the mRNA and protein levels, with greater change at transcript levels. The dashed line represents levels in control cells. Error bars represent SEM. p value is < 0.005; N.S. = not statistically significant; * = statistically significant. All experiments were done in triplicate (n = 3). and CD9 protein for HuR over-expression. There is a more pronounced knock-down, however, in CD9 mRNA in MCF-7 cells with reduced HuR levels. The results of HuR shRNA knock-down in MCF-7 cells were as expected, but opposite of those seen for MB-231 cells. Steady-state mRNA levels of CD9 and CALM2 mRNAs decreased, consistent with the hypothesis that HuR generally stabilizes its mRNA targets. One possible explanation of these disparate results is different levels of total cellular or cytoplasmic HuR. We performed nuclear and cytoplasmic fractionation (Additional File 3, Figure S3). These results demonstrated modest (approximately 10%) greater cytoplasmic levels of HuR in MB-231 cells as compared to MCF-7. The total cellular HuR levels are very similar for both MB-231 and MCF-7 cells. Taken together, these results indicated that HuR appeared to differentially regulate the same mRNAs in a manner dependent upon the cellular milieu.