In transwell co-cultures, the mean percentage of MCF10AT cells labeled by BrdU (i.e., BrdU labeling index) was decreased by 20% in co-culture with NAF (p = 0.011). The NAF utilized

were derived from three different individuals. In direct co-cultures, the mean reduction in BrdU labeling by the same three NAF was 46% (p < 0.001) (Fig. 1). There was variability among the three NAF in their ability to inhibit proliferation of MCF10AT, particularly this website in direct contact co-cultures. The greater reduction in proliferation of MCF10AT in direct versus transwell co-culture was significant (p = 0.04) (Fig. 1). These results indicate that inhibition of epithelial growth by NAF is mediated by a mixture of direct-contact/insoluble and soluble factors.

Therefore, we selected differentially expressed genes from the microarray analysis encoding both soluble and matrix-bound, insoluble molecules for validation by quantitative, real-time PCR (QRT). Fig. 1 Proliferation of MCF10AT in 3D Akt inhibitor direct and transwell co-cultures with NAF. Direct and transwell 3D (i.e., in Matrigel) co-cultures of MCF10AT cells with each of three NAF from different individuals were prepared. BrdU labeling of MCF10AT cells was counted by flow cytometry. Each NAF (i.e., NAF1, NAF2 and NAF3) suppressed proliferation of co-cultured MCF10AT cells to some extent in transwell co-cultures, and two of the three NAF (i.e., NAF1 and NAF3) suppressed proliferation of MCF10AT in direct co-cultures. When comparing the overall reduction in proliferation of Adenosine triphosphate MCF10AT induced by the three NAF in all transwell

co-cultures combined (n = 10, checkered bar) to MCF10AT grown without co-cultured NAF (black bar), the decrease in proliferation was significant (p = 0.011). Similarly, the overall decrease in proliferation induced by the three NAF in all direct co-cultures combined (n = 14, checkered bar) compared to MCF10AT 4SC-202 monocultures (black bar) was significant (p < 0.001). However, the degree of suppression was significantly greater in direct than transwell co-cultures (p = 0.04). Data are normalized to corresponding MCF10AT monocultures. Mean and standard error are shown Expression of a Subset of Differentially Expressed Genes was Confirmed by Real-Time PCR We selected eight genes from the list of 420 differentially expressed genes in NAF and CAF for validation by QRT (Fig. 2a, Supplemental Tables 1 and 2). The primary criterion for selecting genes for validation was that they encoded a secreted protein, either soluble or matrix-bound, that was known to regulate cell growth, migration, invasion and/or ECM remodeling.