Extended Data Fig. 7: NuMA promotes the enrichment of TDP1 at damaged chromatin.

(a) Top; The knockdown efficiency of NuMA was analysed by immunoblotting using anti-NuMA antibodies. Bottom; The expression levels of GFP–TDP1 was measured in cells used in photo-bleaching experiments at time 0 (sec). Error bars (± s.e.m.), n = 3 biological replicates. (b) MRC5 cells were plated onto glass-bottom dishes and co-transfected with GFP-TDP1 and siSCR or siNuMA. Cells were pre-incubated with DMSO or olaparib. Cells expressing similar total GFP signal were locally irradiated with an ultraviolet A laser (405 nm), and GFP–TDP1 accumulation at the site of damage was quantified for the indicated time points. Scale bar-5 µm. (c) Data are plotted as the average percentage fluorescence (arbitrary units) in micro-irradiated tracks ± s.e.m from ∼30 cells measured, n = 3 biological replicates (two-sided, unpaired t-test). (d) Lysates of MRC5 cells transfected with siSCR or siNuMA and siTDP1 either individually or in combination was analysed by immunoblotting, n = 3 biological replicates. (e) MRC5 cells transfected as in (d), treated with H₂O₂ and recovered at indicated time points. Representative violin plot showing spread of comet tail moments from 150 cells, n = 3 biological replicates. (f) Bars represent percentage breaks remaining after removal of H₂O₂ and recovery in complete medium. Error bars (±s.e.m.), n = 3 biological replicates (two-sided, unpaired t-test). (g) Survival of MRC5 cells transfected as in (d) was compared using the indicated doses of H₂O₂. Results are presented on a semi-log scale and represent the average of three biological replicates ± s.e.m. ns - not significant (two-sided, unpaired t-test).