Journal: Nucleic Acids Research

Article Title: The nuclear structural protein NuMA is a negative regulator of 53BP1 in DNA double-strand break repair

doi: 10.1093/nar/gkz138

Figure Lengend Snippet: 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 H 2 O 2 (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 (H 2 O 2 )). In the right panel (H 2 O 2 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-53BP1 ct (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.

Article Snippet: The shRNA vectors targeting human NuMA were purchased from Origene (TR311065).

Techniques: Diffusion-based Assay, Control, MANN-WHITNEY, Immunoprecipitation, Western Blot, Membrane, Expressing, Comparison, Immunostaining, Transfection

Journal: Nucleic Acids Research

Article Title: The nuclear structural protein NuMA is a negative regulator of 53BP1 in DNA double-strand break repair

doi: 10.1093/nar/gkz138

Figure Lengend Snippet: 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-53BP1 ct 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-53BP1 ct 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.

Article Snippet: The shRNA vectors targeting human NuMA were purchased from Origene (TR311065).

Techniques: Expressing, Western Blot, Transfection, Immunostaining, Irradiation, Whisker Assay, Flow Cytometry, MANN-WHITNEY, Phospho-proteomics, Mutagenesis

Journal: Nucleic Acids Research

Article Title: The nuclear structural protein NuMA is a negative regulator of 53BP1 in DNA double-strand break repair

doi: 10.1093/nar/gkz138

Figure Lengend Snippet: NuMA negatively regulates 53BP1 function. ( A ) CSR in CH12F3-2 B cells stably expressing scrambled and 53BP1 shRNA, or nucleofected to express GFP, GFP-NuMA, and GFP-NuMA(S395A) (mean ± SEM; n ≥ 3, ANOVA and Tukey). Representative flow cytometry contour plots are shown for cell nucleofected with GFP and GFP-NuMA constructs. 53BP1 silencing was verified by western blot (right). ( B) Chromosomal aberrations (radials + fusions) in BRCA1-null SUM149 cells transfected with nontargeting or 53BP1-targeting siRNA, GFP, or GFP-NuMA. Cells were treated with olaparib (0.5 μM; 24 h). n = 40–120 cells from ≥3 replicates; statistical analysis with ANOVA and Tukey. Representative images of metaphases are displayed, with arrowheads indicating aberrations. Western blot (right) verified 53BP1 silencing and GFP-NuMA expression (upper band). ( C ) Quantification of NHEJ in U2OS cells with a stably integrated NHEJ-GFP reporter (left; mean ± SEM; Student's t -test). The fraction of cells expressing mCherry or mCherry-NuMA among GFP-positive cells is shown in the cross-ruled graph, whereas cell cycle distribution is shown on the right.

Article Snippet: The shRNA vectors targeting human NuMA were purchased from Origene (TR311065).

Techniques: Stable Transfection, Expressing, shRNA, Flow Cytometry, Construct, Western Blot, Transfection

Journal: Oncotarget

Article Title: Identification and validation of AIB1 and EIF5A2 for noninvasive detection of bladder cancer in urine samples

doi: 10.18632/oncotarget.9406

Figure Lengend Snippet: Highly concordant results between replicate measurements of AIB1 ( A1 , B1 ), EIF5A2 ( A2 , B2 ), and NMP22 ( A3 , B3 ) were obtained in the training and independent validation sets.

Article Snippet: ELISA kits of human AIB1 (SEE798Hu) and human NMP22 (NUMA1: SEC332Hu) were purchased from USCN Life Science (Wuhan, China).

Techniques: Biomarker Discovery

Journal: Oncotarget

Article Title: Identification and validation of AIB1 and EIF5A2 for noninvasive detection of bladder cancer in urine samples

doi: 10.18632/oncotarget.9406

Figure Lengend Snippet: The protein levels of AIB1 ( A1 , B1 ), EIF5A2 ( A2 , B2 ), and NMP22 ( A3 , B3 ) are substantially elevated in BCa urine samples when compared with the non-cancer urine samples in the training and independent validation sets ( p < 0.0001 for both). The results in all groups are presented as the mean and SD. Significance ( p < 0.05) was assessed using the Mann–Whitney U test.

Article Snippet: ELISA kits of human AIB1 (SEE798Hu) and human NMP22 (NUMA1: SEC332Hu) were purchased from USCN Life Science (Wuhan, China).

Techniques: Biomarker Discovery, MANN-WHITNEY

Journal: Oncotarget

Article Title: Identification and validation of AIB1 and EIF5A2 for noninvasive detection of bladder cancer in urine samples

doi: 10.18632/oncotarget.9406

Figure Lengend Snippet: Association of urinary levels of the three markers with selected patient characteristics

Article Snippet: ELISA kits of human AIB1 (SEE798Hu) and human NMP22 (NUMA1: SEC332Hu) were purchased from USCN Life Science (Wuhan, China).

Techniques: Biomarker Discovery

Journal: Oncotarget

Article Title: Identification and validation of AIB1 and EIF5A2 for noninvasive detection of bladder cancer in urine samples

doi: 10.18632/oncotarget.9406

Figure Lengend Snippet: Comparisons of the diagnostic performance by the model and individual AIB1, EIF5A2 or NMP22 in the training cohort A. and independent validation set B. .

Article Snippet: ELISA kits of human AIB1 (SEE798Hu) and human NMP22 (NUMA1: SEC332Hu) were purchased from USCN Life Science (Wuhan, China).

Techniques: Diagnostic Assay, Biomarker Discovery

Journal: Oncotarget

Article Title: Identification and validation of AIB1 and EIF5A2 for noninvasive detection of bladder cancer in urine samples

doi: 10.18632/oncotarget.9406

Figure Lengend Snippet: Logistic regression analysis of biomarkers in voided urine of the training and independent validation sets

Article Snippet: ELISA kits of human AIB1 (SEE798Hu) and human NMP22 (NUMA1: SEC332Hu) were purchased from USCN Life Science (Wuhan, China).

Techniques: Biomarker Discovery