Extended Data Fig. 6: NuMA C-terminal tail or a mitotic separation-of-function mutant promotes SSBR.

(a) Schematic of the truncation mutants generated from NuMA-FL with the C-terminal tail (aa 1700-2115) designated as globular domain (GD) and the microtubule-binding domain (aa 1866-1936) as MD. (b) HEK293 cells were co-transfected with full-length or different truncations of NuMA as in (a), alongside myc-TDP1. GFP-immunoprecipitants were analysed by immunoblotting, n = 3 biological replicates. (c) Immunoblotting of MRC5 cells transfected with siSCR or siNuMA followed by complementation with targeting resistant GFP-tagged NuMA-FL, NuMA-GD, or EV. (d) Representative violin plot showing spread of comet tail moments from 150 MRC5 cells from 3 biological replicates transfected as in (c), post-H₂O₂ treatment and recovered at indicated time points (two-sided, unpaired t-test). (e) Bars represent percentage breaks remaining after removal of H₂O₂ and recovery in complete medium from (d). Error bars (±s.e.m), n = 3 biological replicates, (two-sided, unpaired t-test). (f) HEK293 cells transfected with either NuMA-FL or NuMA-MD followed by nocodazole treatment before harvest. Representative image showing NuMA (green), α-tubulin (red), nuclei (DAPI/blue), n = 6 biological replicates, Scale bar, 10 μm. (g) The percentage of cells with NuMA at the mitotic poles was quantified. Error bars (±s.e.m.), n = 100 cells from 6 biological replicates (two-sided, unpaired t-test). (h) Immunoblotting of GFP-immunoprecipitates from HEK-293 cells transfected with GFP-empty, NuMA-FL or NuMA-MD, alongside myc-TDP1, and treated with H₂O₂, n = 3 biological replicates. (i) Immunoblotting of lysates from MRC5 cells transfected with siSCR or siNuMA and complemented with targeting-resistant GFP-tagged NuMA-FL, NuMA-MD, or EV. (j) MRC5 cells were transfected as in (i), treated with H₂O₂ and recovered in media at the indicated time points. Representative violin plot showing spread of comet tail moments from 150 cells from 3 biological replicates (two-sided, unpaired t-test). (k) Bars represent percentage strand 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).