The F-actin circular structures we observe closely resemble the CD44-rich podosome rosettes described in the literature,. These structures contain MMPs and are sites of collagen-directed matrix degradation. It is likely that the loss of CD44 reactivity from these sites reflect localized CD-44 shedding, which is dependent on MMPs and promotes cancer cell migration and invasion. While a detailed analysis of these ��podosome-like���� structures is beyond the scope of this paper, we have evidence of increased expression of the mRNA for EMT molecules, such as collagen and MMPs, when the levels of LGR5 are reduced. These results are consistent with the hypothesis that collagen and MMPmediated CD44 shedding is responsible for the observed selective loss of CD44 reactivity from F-actin-rich structures when LGR5 levels decrease. There is no known role for LGR5 in any well-defined biochemical pathway or biological process, impeding a direct assessment of Selumetinib changes driven by ICI 182780 altered LGR5 levels. To assess the pathways affected by LGR5 we performed expression profiling using pathway-directed arrays. Given the important role of LGR5- expressing cells in the intestine, we concentrated on three pathways which are of paramount importance for intestinal homeostasis and carcinogenesis, wnt, notch and EMT, using pathway-specific PCR arrays. Expression patterns of pathwayspecific genes in LIM1899 and LIM 1215 cells after knockdown of LGR5 by RNAi, and in LIM 1899 cells after overexpression of LGR5, were compared to those of parental cell lines either untransfected or transfected with empty vectors. To maximize the robustness of the analysis, even at the cost of significance levels, we analysed both stable and transient transfectants as replicates. Altered gene expression was considered specific to LGR5 modulation only if up-or down-regulation was higher than 2-fold relative to the control cells and these changes showed opposite trends in the LGR5 knockdown and LGR5 overexpressing cells. Genes which changed in similar way in both sets were discarded from the analysis. Tables 1, 2 and 3 list, in alphabetical order, the genes specifically affected by LGR5 modulation in the Wnt, Notch and EMT pathways, respectively. Many genes were differentially expressed between control and LGR5 up- or down-regulated cells, but were not significantly altered in the other experimental set: these genes are not included in Tables 1, 2, 3. Our analysis may thus be biased against genes already maximally regulated by the endogenous LGR5 levels. Tables 1, 2, 3 report the results for the LIM1899 cell lines, since we had both knockdown and overexpression samples for this cell line; the genes affected similarly by LGR5 knockdown in LIM1215 are indicated by asterisks. A full report of the analysis can be viewed in Tables S1, S2, S3, S4, S5, S6 and Figures S8, S9, S10.