binding protein 1 53bp1  (Bio-Techne corporation)

Journal: Cancers

Article Title: 53BP1 Accumulation in Circulating Tumor Cells Identifies Chemotherapy-Responsive Metastatic Breast Cancer Patients

doi: 10.3390/cancers12040930

Figure Lengend Snippet: Detection of 53BP1 in CTCs from MBC patients in the DETECT IV trial. ( a ) Selection of MBC patients with CTC-positivity (HER2-) and primary TNBC or primary HER2-, HR+ breast cancer for Eribulin monotherapy and blood sample collection. Blood samples were drawn before therapy (baseline visit), four, twelve, and 24 weeks after the start of Eribulin treatment (1st, 2nd, and 3rd treatment visit) as well as one year after treatment initiation or during premature termination due to disease progression (final visit). The numbers of patients recruited at the different visits are indicated by black columns, the numbers of CTC+ and CTC- patients among them by red and grey columns, respectively. Note that not all of the patients described in entered the study during the baseline visit but during later visits which explains the differences in patient numbers. ( b ) Evaluation of 53BP1 signals in CTCs. Following EpCAM-based CTC enrichment and immunofluorescence microscopic imaging (Cellsearch ® ), CTCs (CK+, CD45-) were enumerated and 53BP1-staining intensity assessed per individual CTC as indicated by representative examples. A 53BP1 score per patient sample was calculated from the percentage of CTCs without and with light, moderate or strong staining. PBMC, peripheral blood mononuclear cell (CK-, CD45+).

Article Snippet: However, the HER2-specific antibody was replaced by Alexa Fluor 488 (AF488)-conjugated anti-53BP1 antibody (NB100-304AF488, 1:50, Novus Biologicals, Centennial, CO, USA).

Techniques: Selection, Biomarker Discovery, Immunofluorescence, Imaging, Staining

Journal: Cancers

Article Title: 53BP1 Accumulation in Circulating Tumor Cells Identifies Chemotherapy-Responsive Metastatic Breast Cancer Patients

doi: 10.3390/cancers12040930

Figure Lengend Snippet: Comparison of CTCs as a function of the HR status in primary tumors or metastases. CTCs from patients with HR+ and HR- primary tumors ( a ) or metastases ( b ) were enumerated and 53BP1 scores determined. Data are shown in box plots with the mean value (dot), median (line), and 95% confidence intervals (CI) (whiskers). For numbers of independent blood samples (N) obtained during different visits, see . * P < 0.05, Kruskal–Wallis test followed by the Mann–Whitney U test. For individual patient scores, see .

Article Snippet: However, the HER2-specific antibody was replaced by Alexa Fluor 488 (AF488)-conjugated anti-53BP1 antibody (NB100-304AF488, 1:50, Novus Biologicals, Centennial, CO, USA).

Techniques: Comparison, MANN-WHITNEY

Journal: Cancers

Article Title: 53BP1 Accumulation in Circulating Tumor Cells Identifies Chemotherapy-Responsive Metastatic Breast Cancer Patients

doi: 10.3390/cancers12040930

Figure Lengend Snippet: Protein expression and nuclear foci formation in breast cancer cell lines. ( a ) Cellular protein levels. 53BP1 and Vimentin band intensities were normalized to loading controls. Values for T47D cell extracts, loaded as a reference on each blot, were set to 1. Mean relative Western blot signals per cell line were calculated from two independent experiments. Box plots in the upper panels show mean values (cross), the median (line), and 95% CI (whiskers) for untreated and for 7d Eribulin-treated non-TNBC (N=4) and TNBC cell lines (N=7). The Kruskal–Wallis test did not reveal significant differences in the case of 53BP1. Representative immunoblots are shown in the bottom panels. Vimentin staining was detected in all TNBC but was negative in all non-TNBC cell lines in which BT20 served as a reference. Actin normalized protein levels are indicated above the 53BP1 and Vimentin signals. ( b ) Focal accumulation of 53BP1 in the nucleus of MBC cell lines. 53BP1 foci were detected by immunofluorescence microscopy in MBC cell lines. The graph presents quantitative data in box plots as in A; ~300–900 nuclei per sample were scored in two independent experiments each, and mean values were calculated for each cell line. *P<0.05, Kruskal–Wallis test, Mann–Whitney U test. Representative immunofluorescence images are displayed in .

Article Snippet: However, the HER2-specific antibody was replaced by Alexa Fluor 488 (AF488)-conjugated anti-53BP1 antibody (NB100-304AF488, 1:50, Novus Biologicals, Centennial, CO, USA).

Techniques: Expressing, Western Blot, Staining, Immunofluorescence, Microscopy, MANN-WHITNEY

Journal: Cancers

Article Title: 53BP1 Accumulation in Circulating Tumor Cells Identifies Chemotherapy-Responsive Metastatic Breast Cancer Patients

doi: 10.3390/cancers12040930

Figure Lengend Snippet: Analysis of genomic integrity as a function of 53BP1 scores of CTCs. The workflow for the determination of the genomic integrity index (GII) following CTC enrichment, staining, single cell sorting, whole genome amplification (WGA), and PCR is schematically drawn on the top. GII values were determined for single CTCs, and average values per sample were calculated. The lower left graph displays CTC numbers with remaining versus loss of genomic integrity for blood samples collected during baseline (GII 1–4, N = 11, and GII 0, N = 9) and treatment visits 1-2 (GII 1–4, N = 5 and GII 0, N = 6). The lower right panel shows 53BP1 scores for these four groups. Box plots show mean values (cross), the median (line), and 95% CI (whiskers). * P < 0.05, Mann–Whitney U test.

Article Snippet: However, the HER2-specific antibody was replaced by Alexa Fluor 488 (AF488)-conjugated anti-53BP1 antibody (NB100-304AF488, 1:50, Novus Biologicals, Centennial, CO, USA).

Techniques: Staining, FACS, Whole Genome Amplification, MANN-WHITNEY

Journal: Cancers

Article Title: 53BP1 Accumulation in Circulating Tumor Cells Identifies Chemotherapy-Responsive Metastatic Breast Cancer Patients

doi: 10.3390/cancers12040930

Figure Lengend Snippet: Longitudinal comparison of PFS curves based on 53BP1 status. Progression-free survival (PFS) curves are based on Kaplan-Meier estimates for MBC patients with CTCs showing a 53BP1 score <50% or ≥50%. ( a ) PFS of patients recruited during the baseline visit. ( b ) PFS of patients recruited during the 1st treatment visit. ( c ) PFS of patients recruited during the final visit.

Article Snippet: However, the HER2-specific antibody was replaced by Alexa Fluor 488 (AF488)-conjugated anti-53BP1 antibody (NB100-304AF488, 1:50, Novus Biologicals, Centennial, CO, USA).

Techniques: Comparison

Journal: Cells

Article Title: CHRDL1 Regulates Stemness in Glioma Stem-like Cells

doi: 10.3390/cells11233917

Figure Lengend Snippet: CHRDL1-depletion radiosensitizes GSCs. ( A ) 53BP1 foci assay 24 h after irradiation of NCH644 shCtrl or NCH644 shCHRDL1 GSCs with the indicated doses. ( B ) Sphere formation of NCH644 shCtrl or shCHRDL1 cells 7 days after seeding of single cells and irradiation as indicated. ( C ) Representative microphotographs of NCH644 shCtrl or NCH644 shCRHDL1 without irradiation (0 Gy) or after irradiation with 6 Gy; scale bar: 200 µm. * p < 0.05; ** p < 0.01; **** p < 0.0001; two-way-ANOVA with Sidak’s multiple comparison tests. ( D ) Sphere formation of NCH644 treated with solvent or recombinant human BMP4 (25 ng/mL) immediately prior to irradiation with a dose of 4 or 6 Gy. Sphere area was determined 7 days after treatment. * p < 0.05; **** p < 0.0001; Kruskal—Wallis test with Dunn’s multiple comparisons test.

Article Snippet: The following primary and secondary antibodies were used: 53BP1 (NP-100-304, Bio-Techne GmbH, Wiesbaden, Germany), 1:1000; Alexa Fluor ® 488 F(ab’)2 fragment goat anti-rabbit IgG (H + L) (Thermo Fisher); 1:500.

Techniques: Irradiation, Comparison, Solvent, Recombinant

Journal: Frontiers in Oncology

Article Title: Inhibition of HSP90 as a Strategy to Radiosensitize Glioblastoma: Targeting the DNA Damage Response and Beyond

doi: 10.3389/fonc.2021.612354

Figure Lengend Snippet: HSP90i by NW457 leads to downregulation of DNA damage response factors, impaired DNA damage repair, and reduced clonogenic survival in response to ionizing irradiation in human glioblastoma cells. (A) Transcriptomic profiling of regulators of the DNA damage response (DDR) in LN229 and T98G cells. mRNA expression levels were determined by qRT-PCR, normalized to a matrix of 3 reference genes (18S rRNA, δ-amino-laevulinate-synthase, and β2-microglobulin), and calibrated to the results of untransformed human astrocytes. For both cell lines, three replicates were analyzed and are displayed as x-fold log2-values. (B) Time course analysis of DDR regulator protein expression in LN229 and T98G cells upon HSP90i by 10 nM NW457. Arrowheads indicate the bands that were used for quantification. Protein levels were normalized to a matrix comprising vinculin and α-tubulin and are depicted as x-fold log2-values compared to the 0 h controls. (C) Immunofluorescence microscopy of γH2AX and 53BP1 DNA damage repair foci in LN229 and T98G cells upon irradiation at 2 Gy ± HSP90i by NW457. Cells were treated with NW457 (10 nM) or DMSO for 24 h, irradiated, and fixed at the indicated times. Cells were stained for γH2AX, 53BP1, and DNA and subjected to deconvolution immunofluorescence microscopy. Scale bar depicts 10 µm. (D) Quantification of DNA damage repair kinetics from (C) . γH2AX and 53BP1 double-positive foci in at least 20 randomly picked nuclei were counted by hand. Individual data points with superimposed means ± 95% confidence intervals are displayed, and overall curve comparison was performed by two-way ANOVA. (E) Clonogenic survival of LN229 and T98G cells upon irradiation at 0–8 Gy ± HSP90i by NW457. Cells were pre-treated with 10 nM NW457 or DMSO for 24 h followed by irradiation at the indicated doses, and colony formation was allowed for 13 d ± continuous NW457 treatment. Individual data points of 4 independent experiments are shown, linear-quadratic regression lines are superimposed, and overall curve comparison was performed by two-way ANOVA.

Article Snippet: Cells were stained with monoclonal mouse anti-γH2AX (Merck Millipore) and polyclonal rabbit anti-53BP1 (Bio-Techne, Wiesbaden, Germany) antibodies diluted in 3% isotonic bovine serum albumin and 0.1% Triton X-100 for 2 h at room temperature.

Techniques: Irradiation, Expressing, Quantitative RT-PCR, Immunofluorescence, Microscopy, Staining, Comparison