Binucleate cells contain nuclear γ-H2AX foci. (A) Binucleate cells were prepared by synchronization and treatment with 25 μM blebbistatin as described above, then stained for the DNA damage marker γ-H2AX. Untreated control cells (top) did not contain any visible γ-H2AX foci, whereas binucleate cells, after released from blebbistatin for 1 h, (bottom) contained γ-H2AX foci that were similar to those of cells treated with H2O2 (middle). Scale bar represents 2.5 μm. (B) Culture was continued for 8 days after release from blebbistatin. Most mononucleate cells lacked γ-H2AX foci (top), whereas approximately 30% of binucleate cells still contained nuclear γ-H2AX foci (bottom). The binucleate cells were also flattened and enlarged, consistent with a senescent-like arrest. Scale bar represents 10 μm.