Figure 6. Larger cells are more prone to DNA damage-induced senescence.

(A) Asynchronous RPE-1 cells were gated for G1 DNA content and sorted into four bins by size using FACS. (B) Sorted cells were replated, cultured in the presence of the DNA damaging agent Doxorubicin (10 ng/ml), and then stained for SA-beta-Gal at the indicated time points to determine the DNA damage-induced senescence dynamics. (C) Large cell size inhibits cell cycle re-entry after G1 arrest or DNA damage to promote senescence. RPE-1 cells were treated with Palbociclib or a low dose of Doxorubicin for 4 days. Then, the drugs were washed out, and the cells were imaged for 4 days to identify cells that re-enter the cell cycle, and cells that remain arrested in a senescent state. Nuclear area was used as a proxy for cell size and cell cycle re-entry was determined using the fluorescent cell cycle phase reporters Cdt1-mKO2 (G1 reporter) and Geminin-mAG (S/G2/M reporter) (Sakaue-Sawano et al., 2008). N = 33 cells for each data point. (D) Protein slope values for all proteins annotated as DNA repair (GO:0006281) and DNA replication (GO:0006260) that were present in our dataset. (E) Immunofluorescence staining of RPE-1 cells treated with 10 ng/ml Doxorubicin for 24 hours against γ-H2AX (red) and 53BP1 (green), with DAPI staining shown in cyan. Scale bar = 10μm. (F) Number of γ-H2AX and 53BP1 loci in RPE-1 cells treated with 10 ng/ml Doxorubicin plotted against the nucleus area, which serves as a proxy for cell size. N = 1265 cells. All the experiments were performed in n = 3 biological replicates. (G) Model of relationships between DNA-related stress, large cell size, and senescence.