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. 2014 Nov 7;9(11):e110844. doi: 10.1371/journal.pone.0110844

Figure 3. Stable tetraploid clones were isolated from diploid D3 and D8 clones after endoreduplication.

Figure 3

A) Shown is the procedure to isolate diploid and tetraploid clones from cells transiently exposed to Nutlin. D3 and D8 were untreated (NT) or treated with 10 µM Nutlin (NUT) for 24 hrs, followed by Nutlin removal for an additional 16 hrs. The cells were then live-stained with Hoechst 33342 (5 µg/ml). Cell sorting was performed on a MoFlo cytometer equipped with a UV excitation wavelength laser. Sorted 2N and 8N cells were plated at low density in normal medium (minus Nutlin) and individual clones isolated. B) Top, the comparison of the DNA profiles between D3 and a representative tetraploid clone isolated from D3. Bottom, comparison of the DNA profiles between a representative diploid clone (D81B) isolated from D8 cells, and a representative tetraploid clone (TD6) isolated from D8 cells. C) Representative metaphase spread from diploid D3 cells and tetraploid T3 cells. The number in the bottom right indicates the number of chromosomes counted. D) FISH analysis with chromosome 17 (Chr 17) and p53-specific probes shows tetraploid (T3) cells contain 4 copies of p53 and Chr 17, while diploid (D3) cells contain 2 copies of p53 and Chr 17.