Figure 6.

Soft agar assays to evaluate the oncogenic function of genes on 8p12 and 11q13.

A. Expression of genes individually and in combination with MYC. Expression of MYC alone in MCF10A gave a few to ~40 colonies per well of a six-well plate. To control for the variability in the number of colonies in MYC expressing cells, all assays with were carried out with MYC alone control. The number of colonies from MYC expressing cells varied little within any one assay. Assays were carried out in triplicate with two different cell lines expressing MYC and each gene generated from two independent infections. Shown are the numbers of colonies per six-well plate (mean and standard deviation) for MCF10A cells stably infected with vector control (empty vector LentiV6 or LPCX) or vectors expressing individual genes, and MCF10A cells stably co-infected with control pBabeH (empty vector) plus appropriate corresponding empty vector for a candidate gene or co-expressing pBabeH-MYC and the selected genes.

B. Expression of genes in combination with GSE56. Expression of GSE56 alone in MCF10A cells produced colonies in soft agar that differed greatly in size, which by western-blotting of clonal cell lines derived from the GSE56-infected MCF10A appeared to result from variable expression levels of GSE56. Therefore, candidate genes were expressed in two different clonal cell lines, one expressing a high level of GSE56 in LXSN (56SN-1) and the other a moderate level (56SN-2). Shown are the numbers of colonies per six-well plate (mean and standard deviation) for MCF10A cells stably infected with vector control (56SN-1 or 56SN-2 and either empty vectors LPCX or LentiV6, V6) or vectors expressing individual genes together with either of the two GSE56-expressing MCF10A clones (56SN-1 or 56SN-2). Cells expressing CCND1 or ZNF703 alone do not form colonies in soft agar (Supplementary Figure 2).