Extended Data Fig. 9: Commitment to senescence in DNA2-deprived cells is mediated by p21 and independent of DNA double-strand breaks.

a, Cytoplasmic (closed arrowheads) and nuclear (open arrowheads) cyclin B1 localisation in RPE-1 OsTIR1 DNA2^dd cells treated or not with DIA (18 h). Mean localisation frequency ± s.d. was determined for n = 3 independent experiments (–DIA, 255 cells; +DIA, 246 cells). Scale bar, 100 μm. b, Effect of p21 siRNA on cell proliferation following DNA2^dd degradation in RPE-1 OsTIR1 DNA2^dd cells (n = 3 independent experiments) and western blot analysis (n = 1) of p21 depletion. p21 siRNA was applied for 24 h before addition of DIA. After three days, nuclei were stained and counted. 3500 cells plated and data normalised to cells treated with non-targeting (NT) control siRNA only. Data presented as mean values ± s.d. c, Effect of p21 siRNA on the increase in nuclear area following DNA2^dd degradation. Experimental set-up as in panel a, but nuclear area measured for the indicated numbers (n) of cells. d, Cell cycle exit override in DIA-treated RPE-1 OsTIR1 DNA2^dd cells induces micronuclei formation. Experiment carried out as in panel b, but micronuclei-positive cells identified and scored. Data presented as mean values ± s.d. for n = 3 independent experiments. e, Determination of the stage of DIA-induced cell-cycle exit in RPE-1 OsTIR1 DNA2^dd cells at which phosphorylation of RPA (pS4/8 on RPA32; a proxy measure for DNA breakage) is detected. Cells were treated 18 h with DIA and stained for cyclin B1. pRPA-positive cells (indicated by white arrowheads) were assessed for cyclin B1. Approximately 5% of cells were positive for pRPA, and cyclin B1 was mostly absent (degraded) in these cells, indicating that any DNA double-strand break formation occurs after commitment to senescence (n = 3 independent experiments). Data presented as mean values ± s.d. Scale bar, 10 μm.

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