Figure 3

Increase of gene copy number of the egfr gene in the breast cancer cell line MDA-MB-468 CD44+/CD24 -/LOW during cell culture. FISH analysis on interphase nuclei was done using an egfr specific probe counterstained with DAPI. The egfr gene amplification of a low EGFR-expressing population (isolated by cell sorting) is increasing from the day of sorting (d = 0) to day 92 of culture. Quantification of in situ hybridization was done by measuring fluorescence areas as single spots were not available in all cases. Here we give an example displaying clearly this increase, as significant higher fluorescence intensities were obtained for cultured cells (T-Test: N1 and N2 = 200; P < 0.0001). Histograms showing the distribution of amplification within the heterogeneous cell populations (green = d0; red = d92) were derived using R and the akima package. Upward diagonals indicate new cells which were not present directly after sorting. Results were confirmed using qPCR.