Fig 2: Immunoblot analysis for the expression of M-phase specific proteins (e.g., phospho-NuMA Ser395 and phospho-H3 Ser10) in H1299 lung cancer (A) and HCC1806 breast cancer (B) treated with DMSO (negative control), EAPC-67, EAPC-70, EAPC-71 (both 10 μM), Colchicine (0.05 μM) and Vinblastine (0.01 μM) for 24 h. Actin stain was used as a loading control.

Fig 3: NuMA interacts with 53BP1 and regulates 53BP1 kinetics. (A) Modeling of GFP-53BP1 diffusion, assuming either a spherical or a rod-shaped structure of the protein. GFP and a fluorescent protein tandem (GFP-mCherry) were used as controls. Time trace simulations are shown with the predicted diffusion times and experimental FCS values (mean ± SEM; n ≥ 15). (B) Diffusion of GFP-53BP1 and GFP measured by FCS. U2OS cells were untreated (control), treated with bleomycin (20 mU/ml, 1 h), or exposed to ionizing radiations (IR, 10 Gy and 30 min recovery). n = 50–60 cells from 2 biological replicates; Mann–Whitney test. (C) Immunoprecipitation of NuMA from U2OS nuclear extracts (N.E.). Nonspecific immunoglobulins (IgGs) were used as controls. Blots were probed for NuMA and 53BP1. Cells were exposed to IR (10 Gy, and 30 min recovery) or treated with H2O2 (1 mM, 10 min) prior IP. Densitometric quantification of 53BP1 pull down is shown on the graph (mean ± SEM; one sample t-test; n = 4 (IR) or 3 (H2O2)). In the right panel (H2O2 treatment), all lanes are from the same immunoblot membrane and were taken with the same exposure. Lanes were reassembled for clarity. (D) FRET efficacy in U2OS cells expressing NuMA fused to mCherry and either GFP (used as control), GFP-53BP1, or GFP-53BP1ct (mean ± SD; n = 20–25 cells from at least two experiments; Kruskal–Wallis and Dunn's multiple comparison test). (E) Colocalization (arrowheads) between 53BP1 foci and bright NuMA features in immunostaining images of S1 cells treated with bleomycin or untreated (control). Scale bar, 5 μm. Overlap is quantified on the graph (mean ± SEM; n = 10 images from two experiments, corresponding to 300 nuclei per condition; Student's t-test). (F) FCS analysis of GFP-53BP1, GFP, and GFP-MeCP2 diffusion. Cells were transfected with nontargeting (NT) or with NuMA-targeting siRNA and were treated with bleomycin as indicated. Statistical analysis with one-way ANOVA and Tukey (GFP-53BP1) or Mann–Whitney tests (GFP and GFP-MeCP2). NuMA silencing was verified by western blot, with lamin B as loading control.

Fig 4: NuMA antagonizes 53BP1 accumulation at DNA damage sites. (A) Left: NuMA silencing and 53BP1 expression analyzed by western blot in HMT-3522 S1 cells transfected with nontargeting (NT) or NuMA-targeting siRNA. Right: IR-induced focal accumulation of 53BP1 in S1 cells transfected with siNT and siNuMA. Confocal images show NuMA and 53BP1 immunostaining in irradiated cells (3 Gy, 2h recovery). The arrowhead points to a cell retaining NuMA expression in contrast to its neighbors. Quantification of 53BP1 foci is presented in the box-and-whisker plot. *P < 0.05 and ***P < 0.0001 (one-way ANOVA and Bonferroni's test; n = 4; 100–300 cells analyzed per condition for each replicate). The inset shows the average number of 53BP1 foci per nucleus in cells that lost (–) or retained (+) NuMA expression within the siNuMA transfection condition. #P < 0.05 (Student's t-test). (B) Cell cycle distribution in S1 cells transfected with NT and NuMA siRNA determined by flow cytometry (mean ± SEM; n = 4; Student's t-test). (C) Accumulation of 53BP1 at ISceI sites in U2OS cells transfected with NT and NuMA siRNA. Cleavage sites were identified using the Lac arrays flanking the ISceI site and 53BP1 was detected by immunostaining. n > 40 cells from three replicates; Student's t-test. (D) Accumulation of GFP-53BP1ct at laser-microirradiated tracks (arrowheads) in U2OS cells expressing mCherry or mCherry-NuMA (insets) or transfected with NT and NuMA-targeting siRNA. The fraction of GFP signal at the tracks is shown on the bar graphs (mean ± SEM, Mann-Whitney test). (E) NuMA phosphorylation at Ser395 (P-NuMA) after DNA damage. Left: western blot analysis. Total NuMA and lamin B were used as loading controls. Right: immunostaining of cells transfected with NT or NuMA siRNA. The arrow points to P-NuMA signals in a cells expressing NuMA. (F) Accumulation of mCherry-53BP1ct after laser-microirradiation in cells expressing GFP, GFP-NuMA, or a nonphosphorytable NuMA mutant [GFP-NuMA(S395A)] (mean ± SEM, Mann-Whitney test). Arrowheads indicate the position of the laser microirradiation tracks. Scale bars, 10 μm.

Fig 5: NuMA expression predicts survival in breast cancer patients. (A) Confocal images of 53BP1 and NuMA immunostaining in invasive ductal carcinoma (IDC). Tissue explants were mock-irradiated (control) or exposed to IR (3 Gy) and left to recover for 1 or 6 h. Arrowheads point to colocalized foci. Scale bar, 10 μm. 53BP1 foci numbers and the overlap between 53BP1 foci and bright NuMA features are quantified in the graphs (mean ± SEM; n = 3 [50–300 foci counted per condition in each sample]; Student's t-test). (B) Correlation between NuMA expression and 53BP1 foci density in irradiated tissues (IDC patients #005–7). Left: representative confocal images. NuMA staining intensity is visualized with a heat map. Center: normalized NuMA intensities are plotted against the densities of 53BP1 foci for each cell nucleus. n = 281; Spearman's p-value is indicated. Right: average foci densities in cells with low NuMA (first quintile) and high NuMA (last quintile) (mean ± SEM; n = 56; Mann–Whitney test). Scale bar, 10 μm. (C) Correlation analysis of NUMA1 and TP53BP1 mRNA expression levels in 1954 breast tumors analyzed by microarray. Data are expressed as log2 normalized signal intensities. Spearman correlation (rs) and the corresponding P-value are shown. (D) Kaplan–Meier plot of distant metastasis-free survival (DMFS) with patients stratified by NUMA1 expression quartiles. The log-rank P-value is shown.