Journal: Cancers

Article Title: Testicular Germ Cell Tumors Acquire Cisplatin Resistance by Rebalancing the Usage of DNA Repair Pathways

doi: 10.3390/cancers13040787

Figure Lengend Snippet: List of the antibodies, commercial sources and dilutions used in this study.

Article Snippet: TP53BP1 , OriGene , TA309918 , 1:1000.

Techniques:

Journal: Cancers

Article Title: Testicular Germ Cell Tumors Acquire Cisplatin Resistance by Rebalancing the Usage of DNA Repair Pathways

doi: 10.3390/cancers13040787

Figure Lengend Snippet: List of the antibodies, commercial sources and dilutions used in this study.

Article Snippet: Other primary antibodies used were as follows: anti-H2AX Ser139 (1:250; Cell Signaling 978), anti-RPA1 (1:250; Santa Cruz Biotechnology B6), anti-RAD51 (1:250 H-92; Santa Cruz Biotechnology), anti-BRCA1 (1:100 D9; Santa Cruz Biotechnology), anti-FANCD2 (1:250; Novus Biologicals, Centennial, CO, USA; NB100-182) and anti-53BP1 (1:250; OriGene, Rockville, MD, USA; ta309918).

Techniques:

Journal: bioRxiv

Article Title: Non-homologous end joining shapes the genomic rearrangement landscape of chromothripsis from mitotic errors

doi: 10.1101/2023.08.10.552800

Figure Lengend Snippet: a) Experimental approach to survey the impact of specific DSB repair pathways on chromosome rearrangements induced by micronucleus formation. Biallelic gene knockouts (KOs) were generated in the background of the CEN-SELECT system in isogenic DLD-1 cells. Y chromosome-specific mis-segregation into micronuclei and rearrangements were induced by treatment with doxycycline and auxin (DOX/IAA). b) Representative examples of metaphase spreads with normal or derivative Y chromosomes. Different types of rearrangements can be visualized by DNA fluorescence in situ hybridization (FISH) using probes targeting the euchromatic portion of the male-specific region (MSY, red) and the heterochromatic region (YqH, green) of the Y chromosome. Rearrangements were induced by 3d DOX/IAA treatment followed by G418 selection. Scale bar, 10 µm. c) Plot summarizing the effect on cell viability after G418 selection ( x-axis ) and rearrangement frequency of the Y chromosome ( y-axis ) for each DSB repair KO clone. d) Proportion of Y chromosomes exhibiting simple or complex rearrangements, as determined by metaphase FISH, following transient centromere inactivation. e) Proportion of inter-and/or intra-chromosomal rearrangements. Data in (d) and (e) represent the mean ± SEM of n = 3 independent experiments for WT, n = 2 KO clones for LIG4 and RAD52 , and n = 3 KO clones for PRKDC , NHEJ1 , TP53BP1 , POLQ , RAD54L , and NBN ; statistical analyses were calculated by ordinary one-way ANOVA test with multiple comparisons. ns, not significant; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001. f) Left: Distribution of Y chromosome rearrangement types as determined by metaphase FISH following 3d DOX/IAA treatment and G418 selection. Data are pooled from 3 independent experiments. Right: Plots depict the mean fold change in each rearrangement type as compared to WT cells. Sample sizes indicate the number of rearranged Y chromosomes examined; data are pooled from 2 or 3 individual KO clones per gene. See also .

Article Snippet: To generate the 53BP1 reporter system, a HaloTag was fused in-frame to the N-terminus of the minimal focus-forming region (FFR, amino acids 1220-1711) of 53BP1 from TP53BP1 cDNA (a gift from Anthony Davis) and cloned into a pBABE-zeo construct (Addgene).

Techniques: Generated, Fluorescence, In Situ Hybridization, Selection, Clone Assay

Journal: bioRxiv

Article Title: Non-homologous end joining shapes the genomic rearrangement landscape of chromothripsis from mitotic errors

doi: 10.1101/2023.08.10.552800

Figure Lengend Snippet: a) Experimental schematic for generating CRISPR/Cas9-mediated biallelic knockout clones. Cleavage at two sgRNA sequences yields a frameshift deletion that can be detected by PCR. b ) Sanger sequencing confirmation of predicted 37 base pair frameshift deletion in the POLQ gene in three independent clones. c) Confirmation of KO clones by immunoblotting. Molecular weight markers are indicated in kilodaltons. d) Representative images of PRKDC KO cells with micronuclei before and after induction with DOX/IAA. Scale bar, 10 µm. e) The percentage of cells with micronuclei with and without 3d DOX/IAA treatment. Data pooled from (left to right): 1,367, 1,711, 2,208, 1,236, 340, 407, 1,554, 1,709, 396, 335, 1,985, 1,769, 935, 295, 1,256, 1,782, 706, and 736 cells. f) Images of metaphase spreads with intact or fragmented Y chromosomes after 4d DOX/IAA treatment. Scale bar, 10 µm. g) Frequency of Y chromosome fragmentation. Only Y chromosome-positive metaphase spreads were scored. Data pooled from (left to right): 168, 267, 113, 226, 81, 141, 162, 162, 148, 147, 184, 213, 79, 123, 120, 186, 215, and 255 metaphase spreads. Bar graphs in ( e ) and ( g ) represent the mean ± SEM from n = 3 independent experiments for WT controls, n = 2 KO clones for LIG4 and RAD52 , and n = 3 KO clones for PRKDC , NHEJ1 , TP53BP1 , POLQ , RAD54L , and NBN ; statistical analyses were calculated by ordinary one-way ANOVA test with multiple comparisons. ns, not significant; *** P ≤ 0.001; **** P ≤ 0.0001.

Article Snippet: To generate the 53BP1 reporter system, a HaloTag was fused in-frame to the N-terminus of the minimal focus-forming region (FFR, amino acids 1220-1711) of 53BP1 from TP53BP1 cDNA (a gift from Anthony Davis) and cloned into a pBABE-zeo construct (Addgene).

Techniques: CRISPR, Knock-Out, Clone Assay, Sequencing, Western Blot, Molecular Weight

Journal: bioRxiv

Article Title: Non-homologous end joining shapes the genomic rearrangement landscape of chromothripsis from mitotic errors

doi: 10.1101/2023.08.10.552800

Figure Lengend Snippet: a) Left: Distribution of Y chromosome rearrangement types as determined by metaphase FISH following continuous passage in DOX/IAA and G418 for ∼30 days. Data are pooled from 2 independent experiments. Right: Plots depict the mean fold change in each rearrangement type as compared to WT cells. Sample sizes indicate the number of rearranged Y chromosomes examined; data are pooled from 2 or 3 individual KO clones per gene. See also . b) Proportion of Y chromosomes exhibiting simple or complex rearrangements, as determined by metaphase FISH, following sustained centromere inactivation. Data represent the mean ± SEM of n = 2 independent experiments for WT, n = 2 KO clones for LIG4 and RAD52 , and n = 3 KO clones for PRKDC , NHEJ1 , TP53BP1 , POLQ , RAD54L , and NBN ; statistical analyses were calculated by ordinary one-way ANOVA test with multiple comparisons. ns, not significant; * P ≤ 0.05. c) Cytogenetic characterization of POLQ KO sub-clones harboring complex Y chromosome rearrangements following sustained centromere inactivation. Scale bar, 10 µm. d) Whole-genome sequencing analyses of POLQ KO sub-clones with complex Y chromosome rearrangements exhibiting oscillating DNA copy-number patterns. For each subclone, sequencing depth (grey dots), copy number information (black lines; top), and inter-mutational distances (bottom) for the mappable regions of the Y chromosome are shown.

Article Snippet: To generate the 53BP1 reporter system, a HaloTag was fused in-frame to the N-terminus of the minimal focus-forming region (FFR, amino acids 1220-1711) of 53BP1 from TP53BP1 cDNA (a gift from Anthony Davis) and cloned into a pBABE-zeo construct (Addgene).

Techniques: Clone Assay, Sequencing

Journal: Chromosoma

Article Title: DNA repair kinetics in SCID mice Sertoli cells and DNA-PKcs-deficient mouse embryonic fibroblasts

doi: 10.1007/s00412-016-0590-9

Figure Lengend Snippet: DSB damage persistence before IR and repair kinetics after IR in Sertoli cells. a Testicular section of a wild-type mouse showing no 53BP1 DSB-indicating foci in Sertoli cells. Sertoli cells are characterized by irregular-shaped nucleus with two blue dots next to the dark spot (the nucleolus) representing the chromocenters that are specific for this cell type. b 53BP1 foci ( red ) in Prdkc scid Sertoli cells ( arrows ). c–f Representative images for IR-induced 53BP1 foci in wild-type and SCID Sertoli cells. g , h γ-H2AX foci in nonirradiated Prdkc scid Sertoli cells ( arrows in G indicate green γ-H2AX foci and arrows in H show co-localized foci). i Irradiated wild-type testis ( arrows show co-localized foci). j Prdkc scid Sertoli cells displaying γH2AX foci co-localized with 53BP1 ( arrows ). S Sertoli, Sc spermatocyte, B type B spermatogonia, A type A spermatogonia, eS early spermatocytes. Scale bars represent 10 μm

Article Snippet: The primary antibodies used were anti-53BP1 rabbit polyclonal (1:400; Acris Antibodies, Herford, Germany) and anti-γ-H2AX mouse monoclonal antibody (1:500, JBW301, Milipore, Germany).

Techniques: Irradiation

Journal: Chromosoma

Article Title: DNA repair kinetics in SCID mice Sertoli cells and DNA-PKcs-deficient mouse embryonic fibroblasts

doi: 10.1007/s00412-016-0590-9

Figure Lengend Snippet: DNA repair kinetics after IR in Sertoli cells of wild-type and Prdkc scid mice. a Percentages of Sertoli cells with foci at different time points after 0.5 Gy gamma-IR. a Average number of 53BP1 foci per Sertoli cell before and at different time points after IR. Fifty cells per mouse were analyzed per time point (three mice each)

Article Snippet: The primary antibodies used were anti-53BP1 rabbit polyclonal (1:400; Acris Antibodies, Herford, Germany) and anti-γ-H2AX mouse monoclonal antibody (1:500, JBW301, Milipore, Germany).

Techniques:

Journal: Chromosoma

Article Title: DNA repair kinetics in SCID mice Sertoli cells and DNA-PKcs-deficient mouse embryonic fibroblasts

doi: 10.1007/s00412-016-0590-9

Figure Lengend Snippet: Telomeres and DSB damage foci at Sertoli cells before and after IR. a – f Representative images form nonirradiated a , b and irradiated d – f wild-type and Prdkc scid testes showing the co-localization of 53BP1 foci at telomeres of TD Sertoli cells ( arrow heads ). g Percentages of 53BP1 foci that overlap (partially or co-localize) with telomere signals before and after IR. S Sertoli, Sc spermatocyte, B type B spermatogonia, A type A spermatogonia, eS early spermatocytes. Scale bars at 10 μm

Article Snippet: The primary antibodies used were anti-53BP1 rabbit polyclonal (1:400; Acris Antibodies, Herford, Germany) and anti-γ-H2AX mouse monoclonal antibody (1:500, JBW301, Milipore, Germany).

Techniques: Irradiation

Journal: Chromosoma

Article Title: DNA repair kinetics in SCID mice Sertoli cells and DNA-PKcs-deficient mouse embryonic fibroblasts

doi: 10.1007/s00412-016-0590-9

Figure Lengend Snippet: Immunofluorescent analysis of different cell cycle phases in 53BP1-stained wild-type, DNA-PKcs −/− and Ku −/− MEF cell lines. Cells were synchronized by serum starvation for 18 h. a Substages of S-phase cells shown according to PCNA ( green ) staining patterns; G 1 and G 2 /M phases are negative for PCNA. b Representative images showing the presence, induction, and disappearance of 53BP1 DSB-indicating foci after IR in wild-type, DNA-PKcs −/− , and Ku −/− MEF cells 0 h, 5 min, 30 min, 1 h, and 7 h post-IR. Note the increase in foci after 7 h of IR in DNA-PKcs −/− cells

Article Snippet: The primary antibodies used were anti-53BP1 rabbit polyclonal (1:400; Acris Antibodies, Herford, Germany) and anti-γ-H2AX mouse monoclonal antibody (1:500, JBW301, Milipore, Germany).

Techniques: Staining

Journal: Chromosoma

Article Title: DNA repair kinetics in SCID mice Sertoli cells and DNA-PKcs-deficient mouse embryonic fibroblasts

doi: 10.1007/s00412-016-0590-9

Figure Lengend Snippet: Radiation-induced 53BP1 foci (RIF) in wild-type and mutant MEF cell lines. a The kinetics of induction and loss of 53BP1 foci in sham-irradiated and X-irradiated wild-type, DNA-PKcs −/− , and Ku −/− MEFs (in G 1 -phase) 5 min, 30 min, 1 h, 3 h, and 7 h post-IR. b Repair kinetics after applying a correction factor based on the FACS analysis and the DI of cell lines. c Repair kinetics in S-phase cells and background foci (at 0 h) were subtracted and foci values were normalized according to the DI. Compared to the wild type ( a ), compared to the Ku −/− ( b ), and compared to DNA-PKcs −/− ( c ). * p < 0.05

Article Snippet: The primary antibodies used were anti-53BP1 rabbit polyclonal (1:400; Acris Antibodies, Herford, Germany) and anti-γ-H2AX mouse monoclonal antibody (1:500, JBW301, Milipore, Germany).

Techniques: Mutagenesis, Irradiation

Journal: Stem Cells International

Article Title: Dual Effects of Hypoxia-Inducible Factors-1 Alpha in Bleomycin-Induced Pulmonary Fibrosis Treated by Human Umbilical Cord Mesenchymal Stem Cells

doi: 10.1155/2021/6658855

Figure Lengend Snippet: Preventive administration of HUCMSCs can effectively attenuate pulmonary fibrosis induced by bleomycin in A549 cells. (a) Results of immunofluorescence staining in A549 cells of the control, CM-pretreatment, CM-treatment, belomycin, belomycin+CM-pretreatment, and belomycin+CM-treatment groups. TP53BP1 are stained in A549 cells with antibodies (red). Nuclei are stained with DAPI (blue). Scale bar = 100 μ m. (b) Results of western blot in the same groups. (c) Results of qPCR in the same groups. Data are presented as mean ± SEM. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01 vs. the control; # p ≤ 0.05, ## p ≤ 0.01 vs. the model.

Article Snippet: The cells were blocked in 5%BSA for 1 h and then incubated with primary antibody at 4°C overnight against TP53BP1 (Boster, BA2878).

Techniques: Immunofluorescence, Staining, Control, Western Blot

Journal: Cell Reports

Article Title: DNA double-strand break-derived RNA drives TIRR/53BP1 complex dissociation

doi: 10.1016/j.celrep.2022.111526

Figure Lengend Snippet:

Article Snippet: TP53BP1 tudor-like region (human recombinant) , Cayman , 14073-100ug-CAY.

Techniques: Purification, Transduction, Control, Virus, Recombinant, In Situ, Software