Progressive Telomere Dysfunction Causes Cytokinesis Failure and Leads to the Accumulation of Polyploid Cells
Figure 5
Artificially induced anaphase bridges engender binucleated polyploid HMEC-hTERT cells.
(A) Immortalized HMECs were treated for one hour with the DNA damage-inducing agent bleomycin (HMEC-hTERT_BLEO) to induce double strand breaks. After drug washout, cells were collected at different timepoints (6, 24, 48 and 72 h after treatment). Non-treated cells (HMEC-hTERT_Control) were also collected in parallel at 0 and 72 hours. Then DNA content was analyzed by flow cytometry and the presence of binucleated cells and anaphase bridges was scored after Texas Red-X phalloidin and DAPI staining. (B) Image showing a binucleated cell (white asterisk) and two nuclei connected by a chromatin bridge (open white arrow). (C) The graph shows the evolution of the percentage of BN and AB on HMEC-hTERT_BLEO (non-patterned bars) and HMEC-hTERT_Control (stripped bars). (D) The table resumes the DNA content values (averaged from three different experiments) resulting from flow cytometry analyses. Standard deviation is shown. (E) Plots resulting from the analysis of the DNA content (Y axes) and the forward light scatter (FLS) (X axes), which allows for the identification of larger cells. Of note, upon drug treatment, a cell subpopulation with a larger area appears (blue dots). An averaged value from three independent experiments is shown. (F) HMEC-hTERT cells were treated with cytochalasin B (HMEC-hTERT_CytB), during 24 hrs before FACS sorting, to artificially obtain binucleated cells. The dot plot shows that the fraction of cells falling in the right gate (in blue) increases upon Cyt-B treatment. Averaged data and standard deviation from three independent experiments is shown.