During normal epithelial organogenesis, mammary epithelial manufacturer Cathepsin G Inhibitor organize into acini, spherical structures containing a hollow lumen . Here, we show that phenotypically normal mammary epithelial cells (S1) in 3D-collagen I cocultures with normal mammary fibroblasts (HMF) arrange into growth-arrested, acini like structures with a central lumen. In vivo, a defined apical¨Cbasal polarity of normal epithelial cells is essential for tissue function . Consistent with literature reports using 3D-cultures in laminin-rich matrices, we demonstrate polarized orientation of the cellular axis in 3D-collagen I gels in coculture with HMF . In contrast, S1 cells in monoculture are characterized by progressive growth, disruption of tissue organization as shown by random distribution of basal (¦Â4-integrin) and apical (golgin-97, ZO-1) polarity markers and destabilization of adherent junctions indicated by diffuse ¦Â-catein expression. Moreover, normal mammary epithelial cells in monoculture display decreased integrin expression, upregulation of the mesenchymal marker vimentin and formation of cellular extensions as frequently observed in carcinoma cells . Collectively, S1 cells growing in the absence of fibroblasts recapitulate many hallmarks of breast cancer such as loss of normal tissue architecture, morphological disorganization and loss of proliferation control .
In summary, our results indicate that the techniques used here allow transcriptome sequencing of 500 or even fewer cells. The amplification works well also with single p53 tumor suppressor structure  but with decreasing cell numbers, individual cells gain influence on the results. Outlier gene expression within a single cell  or stochastic gene expression effects  might overwhelm average expression e.g. of the cancer stem cell population. To avoid potential cell number-dependent bias in the amplification process, we recommend to use at least 100 cells and to keep the number of cells for RNA isolation constant for all analyses. Appendix A. Supplementary material Supplementary data 1. Help with PDF filesOptionsView in workspaceDownload file (2256 K) Supplementary data 2. Help with PDF filesOptionsView in workspaceDownload file (180 K) Supplementary data 3. Help with PDF filesOptionsView in workspaceDownload file (370 K) Supplementary data 4. Help with PDF filesOptionsView in workspaceDownload file (303 K)
The Gly382Asp variant is present in the C-terminal domain of MUTYH which shares homology with MutT. MutT hydrolyzes 8-oxodGTP into 8-oxodGMP thereby preventing its incorporation into DNA ,  and . In MUTYH this domain has been shown to be important for 8-oxoG recognition ,  and . When the corresponding E. coli MutY Gly253Asp variant was examined, it showed a loss of affinity to duplexes that had 8-oxoG rather than G which was similar to results observed with E. coli MutY that was truncated for the C-terminal domain . The Gly253Asp variant of E. coli MutY also showed an 85% Beta-Amyloid solubility in glycosylase activity and failed to complement the mutY mutator phenotype of E. coli. In single turnover experiments the MUTYH Gly382Asp variant exhibited about 30¨C40% the activity of wild type and was able to partially suppress the spontaneous mutation frequency observed in E. coli mutants . Other studies showed glycosylase levels to range from 15% to 50% , ,  and  of wild type levels. In keeping with the in vitro studies, it was reported that MAP patients with homozygous Gly382Asp mutations and heterozygous Tyr165Cys/Gly382Asp mutations had a milder phenotype than homozygous Tyr165Cys patients .