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. Author manuscript; available in PMC: 2011 Apr 2.
Published in final edited form as: Cell. 2010 Apr 2;141(1):81–93. doi: 10.1016/j.cell.2010.01.031

Figure 6. Re-establishment of cell division cycles after tetraploidization.

Figure 6

(A) Schematics of the experimental approach. H2B-GFP expressing cells POT1aF/−POT1bF/F MEFs transduced with POT1a under the control of the Tet-Off inducible promoter were treated with Cre and cloned. Data on one clone (#19) is shown. After 7 days of treatment with doxycycline (doxy) to repress POT1a, cell line #19 was stained with Hoechst and FACS-sorted for DNA content. G1 diploid cells (2N peak) and G2 tetraploid cells (8N peak) were separated, plated in the absence of doxyxycline and monitored by time lapse imaging.

(B) Induction of polyploidy after treatment with doxycycline. Clone #19 described in (A) was analyzed by FACS (PI staining) after 7 days in doxycycline.

(C) Reversible expression of POT1a and silencing of the DNA damage signal. Immunoblotting for the indicated proteins in cell line #19 is shown after 3 or 5 days of doxycycline treatment and 5 days after its removal (day 12) in sorted diploid and tetraploid cells.

(D) Cell divisions of diploid and tetraploid cells derived from clone #19 after re-expression of POT1a. Time lapse imaging of diploid and tetraploid cells expressing H2B-GFP was started 24 hours after doxyxcycline removal. Phase contrast and GFP-fluorescent images were taken every 15 minutes (Movie S8). Selected time points are shown. Arrows highlight a cell performing multiple cell divisions during the imaging session. Insets show evidence for multipolar mitoses.