Fig 2: The chromatin response to DNA damage is altered in cells silencing NuMA. (a) Texture descriptors extracted from confocal images of DAPI-stained nuclei from mock-irradiated (control) or irradiated (3 Gy) cells. The descriptors were condensed to a three-dimensional composite feature space with principal component analysis. Each point on the graph represents one nucleus. Data are from four biological replicates. High specificity, sensitivity and accuracy values indicate separation of the two cell populations. (b) DAPI texture analysis performed as in A in cells transfected with NuMA siRNA or with nontargeting siRNA. Irradiated cells were left to recover for 30 min or 2 h. (c) Measurement of cleaved (+I-SceI) and uncleaved LacO array size in U2OS cells treated with NuMA siRNA or with nontargeting siRNA. Images show LacO arrays labeled with LacR-CFP and γH2AX (used to verify presence/absence of cleavage). Values in the graph represent mean ± SEM (n ∼ 50 cells from three experiments; *P < 0.05, ANOVA and Tukey's multiple comparison test). Scale bars, 50 μm (b) and 1 μm (c).

Fig 3: NuMA interacts with proteins and nucleic acid components of ribosomal biogenesis. S1 cells were cultured for 8 days with complete medium followed by 2 days without EGF to induce proliferation arrest; T4–2 cells were cultured for 6 days. (A) Immunoprecipitation (IP) of nuclear extracts from S1 and T4–2 cells with NuMA antibodies (NuMA) or with non-specific immunoglobulins (IgG), followed by western blot analysis of the input and immunoprecipitated samples using NuMA and NM1 antibodies. (B) Immunoprecipitation of nuclear extracts from S1 cells followed by western blot analysis for NuMA and RNA polymerase I (RNA pol I). (C) ChIP from T4–2 cells with NuMA antibody (n = 4) or RNA Polymerase I antibody (n = 2), followed by RT-qPCR for coding (pro-1, H4, H8, H13) and non-coding (H18) regions of rDNA. The drawing shows the organization of the rDNA gene (IGS = intergenic sequence). Data are normalized to those obtained with IgG control (see ‘Materials and Methods’ section). (D) RNA immunoprecipitation fromT4–2 cells with NuMA antibody or with non-specific IgG of nuclear extracts and soluble extracts (the latter were treated with 50 μg/ml cycloheximide [CH]). Both the DNAse treated non-reverse transcribed RNA (nonRT RNA) and the DNAse treated reverse-transcribed cDNA (cDNA) samples were subjected to PCR using primers specific for human 18S and 28S rRNAs (n = 2). (E and F) Immunoprecipitation of nuclear extracts from S1 cells followed by western blot analysis for NuMA, RPL26 and RPL24.

Fig 4: Silencing NuMA and treatments with actinomycin D and H2O2 lead to p27kip1 increase unless it is already elevated. MCF10A WT-p53 cells and p53-null MDA-MB-157 cells were treated with either siRNA against NuMA (siNuMA) or non-targeting siRNA (siNT) and left in culture for 10 (MCF10A) or eight (MDA-MB-157) days, and/or with 0.08 μg/ml actinomycin D (Act D) or control DMSO for 4 h on day 10 (or 8), and/or with 250 μM of H2O2 for 4 h on day 10 (or 8). (A and C). Percentage of cells with perinucleolar caps of fibrillarin indicative of nucleolar stress (n = 3; 150 nuclei analyzed per condition) in MCF10A (A) and MDA-MB-157 (C) populations. (B and D). Levels of FUrd staining for nascent rRNA (nucleolus) following normalization to nucleoplasmic staining intensity (nascent mRNAs) under the different treatment conditions (n = 3; 100 nuclei analyzed per condition). Shown is a representative western blot of NuMA expression upon siNuMA treatment in MCF10A cells (B) and MDA-MB-157 cells (D). (E and F). Western blot and accompanying quantification (following standardization with lamin B and compared to the first band of each western blot) for p27kip1, following Act D or H2O2 treatment in the presence (siNuMA) or absence (siNT) of siRNA against NuMA; lamin B is used as loading control in MCF10A (E) and MDA-MB-157 (F) cells. (G). Western blot for p53, NuMA, p27kip1 and loading control lamin B in untreated S1, MCF10A and MDA-MB-157 cells.

Fig 5: NuMA is present in the nucleolus of mammary epithelial cells. (A) Immunoblots for NuMA, ribosome-associated proteins fibrillarin and RPL26, cytoplasmic marker α-Tubulin and nucleoplasmic 53BP1 in total protein extracts (Total) and purified nucleoli fraction (Nucleoli) of T4–2 cells. (B). Electron micrographs of growth-arrested S1 cells (day 10) incubated with non-specific immunoglobulins (control) or with NuMA antibodies (NuMA) revealing the localization of NuMA (some of the gold particles [black dots] are indicated by arrows in the inset). A drawing illustrates the different nucleolar subcompartments. (C) Quantification of the distribution of NuMA gold immunolabeling particles within the FC and DFC/GC regions of the nucleoli as determined by the number of NuMA gold particles measured per area of FC and DFC/GC regions from the electron micrographs. The error bars on the scattered plot represent standard deviation. A total of eight nucleoli [areas ranging from ∼0.15 to 1 μm2] were analyzed with four nuclei containing one nucleolus per nucleus, three nuclei containing two nucleoli per nucleus and one nucleus containing three nucleoli; FC, fibrillar center; DFC, dense fibrillar center; GC, granular center.