Figure 4

Clonal changes in KH39, Δ3/KH39 and #3/KH39. A. Dual-color FISH defines chr3 constitution (see explanation to figure 2A and 2B) for c7 in the #3/KH39, 9 weeks of in vitro propagation, and for c9 and c10, representing single cell colonies of the #3/KH39. Marker chromosomes are shown magnified. Orange text box with clone number (c7, c9): clones without supernumerary 3p21 copy. These have two RP6-123I13 signals (red arrows) per metaphase. Yellow text box: clone (c10) with one supernumerary 3p21 copy. It shows three RP6-123I13 signals per metaphase. B. Chr3 constitution for each clone includes N (cytogenetically normal chr3) and M, M1, M2 ... M7 (different marker chromosomes). Underlined: the present or lost chromosome had 3p21 FISH signal. C-F. Percentages of metaphases representing a particular clone at 0, 3 and 9 weeks of propagation; in all analyzed SCID tumors and in 10 single cell colonies (not done for KH39 and Δ3/KH39, "nd"). The tumors that derived from #3/KH39 and Δ3/KH39 have been analyzed previously [8]. Orange, yellow and green shading: expanded clones with no, 1 and 2 supernumerary 3p21 copies, respectively, compared to KH39. The term "expanded clone" refers to a clone that appeared at the given time or its representation was not decreased (ratio >0.8) compared to the previous time point. No shading: decreased representation compared to previous time point. Empty: clone is not present in the sample. Note that chr3 fragments, which were carried by N and M1, were lost in more than one clone expanded during growth ("lost chr3 segments"), whereas ones included into M, M3, M4, M5, M6 and M7 were retained as extra chr3 copies in the expanded clones ("retained chr3 segments").