Journal: International Journal of Molecular Sciences

Article Title: p53 Controls Meiotic Prophase Progression and Crossover Formation

doi: 10.3390/ijms23179818

Figure Lengend Snippet: The absence of p53 alters CO formation. ( A ) Quantification of the number of MLH1 foci per spermatocyte at the pachytene stage. Horizontal lines represent means ± SD. Means (±SD) are indicated above the plot, and (*) indicates statistically significant p < 0.05 for the Mann-Whitney test. The number of cells counted per genotype ( n ) is also indicated; ( B , C ) Representative pachytene spermatocytes are shown from wild-type and p53 −/− mice, stained against MLH1 (red), SYCP3 (green), and DAPI (blue). The scale bar in ( C ) represents 10 µm and applies to all panels; ( D ) Bar graph shows MLH1 foci mean for each individual mouse ( N = 3). Means (±SD) are indicated above the plot, (*) indicates statistically significant p < 0.05 for t -test; ( E ) Graph shows MLH1 foci distribution as a cumulative fraction along the SC length for bivalents with a single MLH1. The X-axis represents the relative length from the centromeric end (left) to the distal telomere (right); ( F ) Graph plots the cumulative fraction of MLH1 foci for bivalents with two MLH1 foci; ( G ) Violin plot represents the distribution of the first and second foci along the relative SC length in chromosomes displaying two MLH1 foci; ( H ) The cumulative fraction of inter-focus distances is shown.

Article Snippet: Primary antibodies used were mouse anti-SYCP3 (Abcam, Cambridge, United Kingdom, 1:400), rabbit anti-SYCP3 (Abcam, Cambridge, United Kingdom, 1:400), mouse anti-γH2AX (Millipore, Burlington, MA, USA, 1:400), guinea-pig anti-H1t (kind gift from M. A. Handel, Jackson Laboratory, Bar Harbor, ME, USA, 1:500), mouse anti-MLH1 (G168-15) (BD Biosciences, Franklin Lakes, NJ, USA, 1:50).

Techniques: MANN-WHITNEY, Staining

Journal: Nature Communications

Article Title: Rad52 competes with Ku70/Ku86 for binding to S-region DSB ends to modulate antibody class-switch DNA recombination

doi: 10.1038/ncomms14244

Figure Lengend Snippet: ( a ) B cells purified from Rad52 +/+ and Rad52 −/− C57BL/6 littermates were stimulated with mCD154 or LPS plus IL-4 (for CSR to IgG1), LPS alone (IgG3), LPS plus IFN-γ (IgG2c) or LPS plus TGF-β, IL-4, IL-5 and anti-δ mAb/dex (IgA). Purified Aicda +/+ and Aicda −/− B cells were stimulated with LPS plus IL-4. After 96 h of culture, the cells were analysed for surface IgG1, IgG3, IgG2c or IgA by flow cytometry. ( b ) Proliferation of Rad52 +/+ and Rad52 −/− B cells labelled with CFSE and stimulated for 72 h with different amounts of LPS or mCD154 plus IL-4. Progressive left shift of fluorescence intensity indicates B220 + B cell division. ( c ) Proliferation of Rad52 +/+ and Rad52 −/− B cells labelled with the cell division tracking fluorochrome CFSE and stimulated by mCD154 plus IL-4 or LPS plus IL-4 for 96 h. CFSE intensity and surface IgG1 expression analysed by flow cytometry. Proportion of surface IgG1 + B cells at each cell division indicated. P -values determined using a paired Student's t -test. Data are from one representative (left panels of each condition) or mean±s.d. of three independent experiments (right panels of each condition). ( d ) Recombinant Sμ–Sγ1 or Sμ–Sγ3 DNAs analysed by digestion–circularization PCR (DC-PCR) using serially twofold diluted HindIII digested and T4 DNA ligase-ligated genomic DNA from Rad52 +/+ or Rad52 −/− B cells after stimulation with LPS or LPS plus IL-4 for 96 h. Gapdh was used as a control for ligation and DNA loading. Data are from one representative of three independent experiments. ( e ) Rad52 +/+ and Rad52 −/− B cells were cultured with LPS or LPS plus IL-4 for 60 h. Aicda , Rad52 , Ku70 , Ku86 , Polθ , germline Iγ1-Cγ1 and Iγ3-Cγ3, circle Iγ1-Cμ and Iγ3-Cμ, and post-recombination Iμ-Cγ1 and Iμ-Cγ3 transcripts analysed by real-time qRT–PCR. Each sample was run in triplicate; expression normalized to Cd79b expression and depicted as relative to the expression in Rad52 +/+ B cells, set as 1. Data are from three independent experiments involving three pairs of Rad52 +/+ and Rad52 −/− mice (mean±s.d.). P values determined using a paired Student's t -test. ( f ) Expression of AID, Rad52, Ku70, Ku86, Polθ and β-Actin proteins in unstimulated Rad52 +/+ and Rad52 −/− B cells or Rad52 +/+ and Rad52 −/− B cells stimulated with LPS plus IL-4 for 72 h were analysed by immunoblotting. Data are from one representative of three independent experiments.

Article Snippet: Single-cell suspensions were prepared from spleens of Rad52 +/+ and Rad52 −/− mice, and stained with PE-anti-B220 monoclonal antibody (clone RA3-6B2, eBioscience, 0.05 μg ml −1 ), fluorescein isothiocyanate (FITC)-anti-CD3 monoclonal antibody (clone 17A2, BioLegend, 0.1 μg ml −1 ), FITC-anti-CD4 (clone GK1.5, BioLegend, 0.1 μg ml −1 ) monoclonal antibody and/or allophycocyanin (APC)-anti-CD8 monoclonal antibody (53-6.7, BD Biosciences, 0.1 μg ml −1 ), Alexa Fluor 647-peanut agglutinin (PNA; Invitrogen) and biotin-anti-CD138 monoclonal antibody (clone 281-2, BD Biosciences, 0.05 μg ml −1 ) followed by FITC-streptavidin (11-4317-87, eBioscience, 0.5 μg ml −1 ) or PE-streptavidin (12-4317-87, eBioscience, 0.1 μg ml −1 ), PE-anti-IgM monoclonal antibody (clone AF6-78, BD Biosciences, 0.1 μg ml −1 ), FITC-anti-IgG1 monoclonal antibody (clone A85-1, BD Biosciences, 0.1 μg ml −1 ), FITC-anti-IgG3 monoclonal antibody (R40-82, BD Biosciences, 0.1 μg ml −1 ), FITC-anti-IgA monoclonal antibody (clone C10-3, BD Biosciences, 0.1 μg ml −1 ) and biotin-rat-anti-mouse IgG2a/c monoclonal antibody (clone R19-15, BD Biosciences, 0.1 μg ml −1 ), followed by APC-streptavidin (550874, eBioscience, 0.5 μg ml −1 ) and 7-AAD (BD Biosciences, 0.25 μg per 1 × 10 6 cells).

Techniques: Purification, Flow Cytometry, Fluorescence, Expressing, Recombinant, Ligation, Cell Culture, Quantitative RT-PCR, Western Blot

Journal: Nature Communications

Article Title: Rad52 competes with Ku70/Ku86 for binding to S-region DSB ends to modulate antibody class-switch DNA recombination

doi: 10.1038/ncomms14244

Figure Lengend Snippet: ( a ) Polθ +/+ and Polθ −/− B cells were stimulated with LPS plus IL-4 (for CSR to IgG1), LPS alone (IgG3), LPS plus IFN-γ (IgG2c) or LPS plus TGF-β, IL-4, IL-5 and anti-δ mAb/dex (IgA). After a 96 h of culture, the B cells were analysed for surface IgG1, IgG3, IgG2c or IgA by flow cytometry. ( b ) Proliferation of Polθ +/+ and Polθ −/− B cells labelled with CFSE and stimulated by LPS plus IL-4 for 96 h. CFSE intensity and surface IgG1 expression analysed by flow cytometry. Proportion of surface IgG1 + B cells at each cell division indicated. P -values determined using a paired Student's t -test. Data are from one representative of three independent experiments. ( c ) Polθ +/+ and Polθ −/− B cells cultured with LPS or LPS plus IL-4 for 60 h. Expression of Polθ , Aicda , germline Iγ1-Cγ1 and Iγ3-Cγ3, circle Iγ1-Cμ and Iγ3-Cμ, and post-recombination Iμ-Cγ1 and Iμ-Cγ3 transcripts analysed by qRT–PCR and normalized to Gapdh transcript, depicted as relative to the expression of each transcript in Polθ +/+ B cells, set as 1. Each sample was run in triplicate. Data are from three independent experiments involving three pairs of Polθ +/+ and Polθ −/− mice (mean±s.d.). P -values determined using a paired Student's t -test. ( d , e ) Polθ +/+ and Polθ −/− littermates were injected with NP 16 -CGG and killed 10 days later. ( d ) Titres of circulating IgM, IgG1 and IgA analysed by enzyme-linked immunosorbent assay (ELISA), expressed as μgeq ml −1 . P -values determined using a paired Student's t -test. Data are from three independent experiments (mean±s.d.). ( e ) Surface IgG1 expression in spleen B220 + PNA hi GC B cells analysed by flow cytometry. Data are from one representative of three independent experiments.

Article Snippet: Single-cell suspensions were prepared from spleens of Rad52 +/+ and Rad52 −/− mice, and stained with PE-anti-B220 monoclonal antibody (clone RA3-6B2, eBioscience, 0.05 μg ml −1 ), fluorescein isothiocyanate (FITC)-anti-CD3 monoclonal antibody (clone 17A2, BioLegend, 0.1 μg ml −1 ), FITC-anti-CD4 (clone GK1.5, BioLegend, 0.1 μg ml −1 ) monoclonal antibody and/or allophycocyanin (APC)-anti-CD8 monoclonal antibody (53-6.7, BD Biosciences, 0.1 μg ml −1 ), Alexa Fluor 647-peanut agglutinin (PNA; Invitrogen) and biotin-anti-CD138 monoclonal antibody (clone 281-2, BD Biosciences, 0.05 μg ml −1 ) followed by FITC-streptavidin (11-4317-87, eBioscience, 0.5 μg ml −1 ) or PE-streptavidin (12-4317-87, eBioscience, 0.1 μg ml −1 ), PE-anti-IgM monoclonal antibody (clone AF6-78, BD Biosciences, 0.1 μg ml −1 ), FITC-anti-IgG1 monoclonal antibody (clone A85-1, BD Biosciences, 0.1 μg ml −1 ), FITC-anti-IgG3 monoclonal antibody (R40-82, BD Biosciences, 0.1 μg ml −1 ), FITC-anti-IgA monoclonal antibody (clone C10-3, BD Biosciences, 0.1 μg ml −1 ) and biotin-rat-anti-mouse IgG2a/c monoclonal antibody (clone R19-15, BD Biosciences, 0.1 μg ml −1 ), followed by APC-streptavidin (550874, eBioscience, 0.5 μg ml −1 ) and 7-AAD (BD Biosciences, 0.25 μg per 1 × 10 6 cells).

Techniques: Flow Cytometry, Expressing, Cell Culture, Quantitative RT-PCR, Injection, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: Rad52 competes with Ku70/Ku86 for binding to S-region DSB ends to modulate antibody class-switch DNA recombination

doi: 10.1038/ncomms14244

Figure Lengend Snippet: ( a ) Rad52 +/+ and Rad52 −/− littermates were injected with NP 16 -CGG and killed 10 days later. Titres of circulating total IgM, IgG1 and IgA, NP 32 -binding and (high affinity) NP 4 -binding IgG1 analysed by enzyme-linked immunosorbent assay (ELISA), expressed as μgeq ml −1 or number of dilutions needed to reach 50% of saturation binding (relative units, RU). ( b ) Rad52 +/+ and Rad52 −/− littermates were injected twice with OVA at day 0 and day 7. Titres of circulating total IgE analysed by ELISA at day 7 (before the second OVA injection) and day 13, expressed as ngeq ml −1 . Each symbol represents an individual mouse, n= 5 or 6 pairs of mice. P -values determined using a paired Student's t -test. ( c ) Surface IgG1 expression in spleen B220 + PNA hi GC B cells or IgA expression in Peyer's patch B220 + PNA hi GC B cells of NP 16 -CGG-injected Rad52 +/+ and Rad52 −/− littermates analysed by flow cytometry. ( d ) Flow cytometry analysis of spleen cells from NP 16 -CGG-injected Rad52 +/+ and Rad52 −/− littermates for the proportion of: B220 + B cells and CD3 + T cells, CD4 + and CD8 + T cells, B220 + PNA hi GC B cells, B220 lo CD138 + plasma cells and viable (7-AAD – ) B220 + B cells. ( e ) Rad52 +/+ and Rad52 −/− littermates were injected with NP 16 -CGG. Ten days after the injection, the mice were injected i.p. with BrdU twice within a 16 h interval and killed 4 h after the last injection. Left panels: proliferating B cells (BrdU-stained B220 + B cells) analysed by flow cytometry; middle and right panels: in vivo cell cycle analysis (quadrant corresponding to the G0/G1, S and G2/M phase of the cell cycle) of B220 + B cells and B220 + PNA hi GC B cells. Data are from one representative of three independent experiments.

Article Snippet: Single-cell suspensions were prepared from spleens of Rad52 +/+ and Rad52 −/− mice, and stained with PE-anti-B220 monoclonal antibody (clone RA3-6B2, eBioscience, 0.05 μg ml −1 ), fluorescein isothiocyanate (FITC)-anti-CD3 monoclonal antibody (clone 17A2, BioLegend, 0.1 μg ml −1 ), FITC-anti-CD4 (clone GK1.5, BioLegend, 0.1 μg ml −1 ) monoclonal antibody and/or allophycocyanin (APC)-anti-CD8 monoclonal antibody (53-6.7, BD Biosciences, 0.1 μg ml −1 ), Alexa Fluor 647-peanut agglutinin (PNA; Invitrogen) and biotin-anti-CD138 monoclonal antibody (clone 281-2, BD Biosciences, 0.05 μg ml −1 ) followed by FITC-streptavidin (11-4317-87, eBioscience, 0.5 μg ml −1 ) or PE-streptavidin (12-4317-87, eBioscience, 0.1 μg ml −1 ), PE-anti-IgM monoclonal antibody (clone AF6-78, BD Biosciences, 0.1 μg ml −1 ), FITC-anti-IgG1 monoclonal antibody (clone A85-1, BD Biosciences, 0.1 μg ml −1 ), FITC-anti-IgG3 monoclonal antibody (R40-82, BD Biosciences, 0.1 μg ml −1 ), FITC-anti-IgA monoclonal antibody (clone C10-3, BD Biosciences, 0.1 μg ml −1 ) and biotin-rat-anti-mouse IgG2a/c monoclonal antibody (clone R19-15, BD Biosciences, 0.1 μg ml −1 ), followed by APC-streptavidin (550874, eBioscience, 0.5 μg ml −1 ) and 7-AAD (BD Biosciences, 0.25 μg per 1 × 10 6 cells).

Techniques: Injection, Binding Assay, Enzyme-linked Immunosorbent Assay, Expressing, Flow Cytometry, Staining, In Vivo, Cell Cycle Assay

Journal: Nature Communications

Article Title: Rad52 competes with Ku70/Ku86 for binding to S-region DSB ends to modulate antibody class-switch DNA recombination

doi: 10.1038/ncomms14244

Figure Lengend Snippet: ( a ) Ku70/Ku86 and Rad52 are recruited to CSR-targeted S region DNA in an AID-dependent manner in B cells undergoing CSR. Aicda +/+ and Aicda −/− B cells were stimulated with LPS or LPS plus IL-4 for 60 h. Cross-linked chromatin was precipitated using rabbit anti-Rad52 antibody or mouse anti-Ku70/Ku86 mAb. Precipitated Sμ, Sγ1 and Sγ3 DNA quantified by real-time quantitative PCR (qPCR); amounts relative to those in Aicda +/+ B cells, set as 1. Each precipitated DNA sample was run as triplicate in qPCR; the average of each triplicate was used as data point for that individual sample. Data are from three independent experiments involving three pairs of Aicda +/+ and Aicda −/− mice (mean±s.d.). P -values determined using a paired Student's t -test. ( b ) Precipitated Sμ and Cμ DNA from Aicda +/+ and Aicda −/− B cells detected by PCR. Data are one representative of three independent experiments. ( c ) Rad52 +/+ and Rad52 −/− B cells were stimulated with LPS or LPS plus IL-4 for 60 h. Chromatin was cross-linked and precipitated using a mouse anti-Ku70/Ku86 mAb. The precipitated Sμ, Sγ1 and Sγ3 DNAs quantified by real-time qPCR; amounts in Rad52 −/− B cells are as relative to those in Rad52 +/+ B cells, set as 1. Each precipitated DNA sample was run as triplicate in qPCR; the average of each triplicate was used as data point for that individual sample. Data are from three independent experiments involving three pairs of Rad52 +/+ and Rad52 −/− mice (mean±s.d.). P -values determined using a paired Student's t -test. ( d ) Rad52 +/+ and Rad52 −/− B cells were stimulated with LPS or LPS plus IL-4 for 60 h. DNA ends were labeled in situ with bio-dUTP using TdT. Chromatin was cross-linked and precipitated using mouse anti-Ku70/Ku86 mAb (left panel) or rabbit anti-Rad52 antibody (right panel). After resuspension, DNA with broken ends was pulled down with streptavidin magnetic beads. Precipitated Sμ, Sγ1, Sγ3 and Cμ DNAs quantified by real-time qPCR (this approach allowed for detection of Ku70/Ku86 or Rad52 bound to DSB-free ends); amounts of precipitated DNA are relative to those in Rad52 +/+ B cells, set as 1 (left panel), or relative to respective input DNA (right panel). Each precipitated DNA sample was run as triplicate in qPCR; the average of each triplicate was used as data point for that individual sample. Data are from three independent experiments involving three pairs of Rad52 +/+ and Rad52 −/− mice (mean±s.d.). P -values determined using a paired Student's t -test. ( e , f ) EMSA using a biotin-labelled double-stranded Sμ DNA probe and ( e ) recombinant human RAD52 and KU70/KU86 (constant amount) proteins at different ratios (1:1, 2:1 and 4:1), or ( f ) nuclear extracts (10 μg protein) from Rad52 +/+ and Rad52 −/− B cells (stimulated with LPS plus IL-4 for 60 h) incubated with mouse anti-Ku70/Ku86 mAb or irrelevant mouse IgG (control). The formation of protein–DNA complexes was shifted by anti-Ku70/Ku86 mAb. Numbers below gel image indicate relative density of protein–DNA complex bands normalized with the density of free probe. Shown is one representative gel of three independent experiments.

Article Snippet: Single-cell suspensions were prepared from spleens of Rad52 +/+ and Rad52 −/− mice, and stained with PE-anti-B220 monoclonal antibody (clone RA3-6B2, eBioscience, 0.05 μg ml −1 ), fluorescein isothiocyanate (FITC)-anti-CD3 monoclonal antibody (clone 17A2, BioLegend, 0.1 μg ml −1 ), FITC-anti-CD4 (clone GK1.5, BioLegend, 0.1 μg ml −1 ) monoclonal antibody and/or allophycocyanin (APC)-anti-CD8 monoclonal antibody (53-6.7, BD Biosciences, 0.1 μg ml −1 ), Alexa Fluor 647-peanut agglutinin (PNA; Invitrogen) and biotin-anti-CD138 monoclonal antibody (clone 281-2, BD Biosciences, 0.05 μg ml −1 ) followed by FITC-streptavidin (11-4317-87, eBioscience, 0.5 μg ml −1 ) or PE-streptavidin (12-4317-87, eBioscience, 0.1 μg ml −1 ), PE-anti-IgM monoclonal antibody (clone AF6-78, BD Biosciences, 0.1 μg ml −1 ), FITC-anti-IgG1 monoclonal antibody (clone A85-1, BD Biosciences, 0.1 μg ml −1 ), FITC-anti-IgG3 monoclonal antibody (R40-82, BD Biosciences, 0.1 μg ml −1 ), FITC-anti-IgA monoclonal antibody (clone C10-3, BD Biosciences, 0.1 μg ml −1 ) and biotin-rat-anti-mouse IgG2a/c monoclonal antibody (clone R19-15, BD Biosciences, 0.1 μg ml −1 ), followed by APC-streptavidin (550874, eBioscience, 0.5 μg ml −1 ) and 7-AAD (BD Biosciences, 0.25 μg per 1 × 10 6 cells).

Techniques: Real-time Polymerase Chain Reaction, Labeling, In Situ, Magnetic Beads, Recombinant, Incubation

Journal: Nature Communications

Article Title: Rad52 competes with Ku70/Ku86 for binding to S-region DSB ends to modulate antibody class-switch DNA recombination

doi: 10.1038/ncomms14244

Figure Lengend Snippet: Rad52 −/− and Rad52 +/+ B cells were transduced with pGFP-C-Ku86-shRNALenti lentiviral vector expressing Ku86 -specific shRNA and GFP, or pGFP-C-scr-shRNALenti lentiviral vector expressing scrambled shRNA and GFP. Rad52 +/+ B cells were activated with LPS for 12 h and transduced with empty pMIG-GFP retroviral vector or pMIG-GFP-Rad52 retroviral vector expressing recombinant Rad52. After lentiviral or retroviral transduction, B cells were cultured for 96 h with LPS plus IL-4. ( a ) B-cell expression of Ku86, Rad52 and β-Actin proteins analysed by immunoblotting. ( b ) Proportions of surface IgG1 + B cells among pGFP-C-scr-shRNALenti or pGFP-C-Ku86-shRNALenti lentiviral vector-transduced (B220 + GFP + ) Rad52 −/− and Rad52 +/+ B cells were analysed by flow cytometry. Data are from one representative of three independent experiments. ( c ) Proportions of surface IgG1 + B cells among empty pMIG-GFP or pMIG-GFP-Rad52 retroviral vector-transduced (B220 + GFP + ) Rad52 +/+ B cells were analysed by flow cytometry. Data are from one representative of three independent experiments.

Article Snippet: Single-cell suspensions were prepared from spleens of Rad52 +/+ and Rad52 −/− mice, and stained with PE-anti-B220 monoclonal antibody (clone RA3-6B2, eBioscience, 0.05 μg ml −1 ), fluorescein isothiocyanate (FITC)-anti-CD3 monoclonal antibody (clone 17A2, BioLegend, 0.1 μg ml −1 ), FITC-anti-CD4 (clone GK1.5, BioLegend, 0.1 μg ml −1 ) monoclonal antibody and/or allophycocyanin (APC)-anti-CD8 monoclonal antibody (53-6.7, BD Biosciences, 0.1 μg ml −1 ), Alexa Fluor 647-peanut agglutinin (PNA; Invitrogen) and biotin-anti-CD138 monoclonal antibody (clone 281-2, BD Biosciences, 0.05 μg ml −1 ) followed by FITC-streptavidin (11-4317-87, eBioscience, 0.5 μg ml −1 ) or PE-streptavidin (12-4317-87, eBioscience, 0.1 μg ml −1 ), PE-anti-IgM monoclonal antibody (clone AF6-78, BD Biosciences, 0.1 μg ml −1 ), FITC-anti-IgG1 monoclonal antibody (clone A85-1, BD Biosciences, 0.1 μg ml −1 ), FITC-anti-IgG3 monoclonal antibody (R40-82, BD Biosciences, 0.1 μg ml −1 ), FITC-anti-IgA monoclonal antibody (clone C10-3, BD Biosciences, 0.1 μg ml −1 ) and biotin-rat-anti-mouse IgG2a/c monoclonal antibody (clone R19-15, BD Biosciences, 0.1 μg ml −1 ), followed by APC-streptavidin (550874, eBioscience, 0.5 μg ml −1 ) and 7-AAD (BD Biosciences, 0.25 μg per 1 × 10 6 cells).

Techniques: Transduction, Plasmid Preparation, Expressing, shRNA, Recombinant, Cell Culture, Western Blot, Flow Cytometry