Journal: International Urology and Nephrology

Article Title: Predictive tissue markers in testicular germ cell tumors: Immunohistochemical expression of MLH1 and REV-7 proteins

doi: 10.1007/s11255-023-03933-2

Figure Lengend Snippet: Association of REV-7 with participants’ demographics and clinical characteristics

Article Snippet: Sakurai et al. used the Anti-MAD2B (REV7) antibody (BD Biosciences, Franklin Lake, NJ, USA), while we used the Anti–REV7 antibody (Abcam, Cambridge, UK).

Techniques: Adjuvant

Journal: Nature Communications

Article Title: REV7 is required for processing AID initiated DNA lesions in activated B cells

doi: 10.1038/s41467-020-16632-8

Figure Lengend Snippet: a CSR levels to IgG1 after LPS/IL4 stimulation at Day 3 and 4. n = 5 for WT and 53bp1 −/− ; n = 4 for Cd19cre and Cd19cre Rev7 fl/fl . b Growth curve of indicated B cells upon LPS/IL4 (left) or LPS (right) stimulation. Cell numbers at Day 4 of four sets of mice are subjected to statistical calculation. n = 4 for all genotypes. c Percentages of apoptotic cell (AnnexinV + PI − ) at Day 4 after stimulating with LPS/IL4 (upper) or LPS (lower) are plotted. n = 4 for WT and 53bp1 −/− ; n = 3 for Cd19cre and Cd19cre Rev7 fl/fl . d Multiple roles of REV7. e End resection of AID-initiated breaks at Sγ1 is shown for indicated B cells. Resection ratio is defined as previously reported . n = 4 for WT; n = 3 for 53bp1 −/− , Cd19cre , and Cd19cre Rev7 fl/fl . f Percentage of cells in G2/M phase is plotted for the indicated genotypes. n = 4 for WT, 53bp1 −/− , and Cd19cre Rev7 fl/fl ; n = 3 for Cd19cre . g Mutation spectrum of C/G in the 5′ Sμ region is shown for both strands of genomic DNA. n = 3 for all genotypes. n , independent mice. Data are represented as mean ± SD in a , b , c , e , f , and g . Two-tailed unpaired t -test was performed for b and one-way ANOVA followed by Dunnett’s multiple comparisons test was performed for a , c , e , f , and g . Data from Rev7 knockout are compared with those from other genotypes. **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05, ns: p > 0.05. P -values and sample sizes are provided in Supplementary Table . Source data are provided as a Source Data file.

Article Snippet: Antibodies for ATM (2873S; Cell Signaling; 1:1000), 53BP1 (NB100-304; NOVUSBIO; 1:1000), RIF1 (ab1213422; Abcam; 1:500), REV7 (A9861; Abclonal; 1:1000), REV1 (sc-393022; Santa Cruz; 1:1000), REV3L (GTX17515; Gene Tex; 1:1000), AID (A16217; Abclonal; 1:1000), MSH2 (ab227941; Abcam; 1:1000), β-actin (AC028; Abclonal; 1:10,000), FLAG (F1804, Sigma; 1:1000), β-Tubulin (A01030HRP; Abbkine; 1:10,000), glyceraldehyde 3-phosphate dehydrogenase (AB2000; Abways; 1:20,000), and Rabbit TrueBlot (18-8816-33; Ebioscience; 1:1000) were used in western blotting.

Techniques: Mutagenesis, Two Tailed Test, Knock-Out

Journal: Nature Communications

Article Title: REV7 is required for processing AID initiated DNA lesions in activated B cells

doi: 10.1038/s41467-020-16632-8

Figure Lengend Snippet: a Survival curves of indicated CH12F3 cell lines upon γ-irradiation (IR), UVC, and cisplatin treatment are plotted as means ± SD at left. Area-under-curve (AUC) are calculated and means ± SEM are compared at the right. In IR treatment, n = 13 for parental CH12F3 cells; n = 12 for Rev7 −/− ; n = 4 for Rev1 −/− and Rev1 Δ9/Δ9 ; n = 9 for the other genotypes. In UVC treatment, n = 4 for parental CH12F3 cells and Rev1 −/− ; n = 6 for Rev7 −/− and Rev3l −/− . n = 3 for the other genotypes. In cisplatin treatment, n = 9 for parental CH12F3 cells; n = 9 for Rev1 −/− ; n = 3 for the other genotypes. CSR rate from IgM to IgA ( b ), end resection level in Sα region ( c ), and percentage of C > G transversion in 5′Sμ region ( d ) are shown for the indicated cells. Cell numbers ( e ) and percentage of apoptotic population ( f ) with/without cytokine stimulation (CIT, w.o.CIT) at Day 3 are showed. Colored points indicate individual knockout clones. In b , n = 5 for parental CH12F3 cells; n = 4 for Rev7 −/− , 53bp1 −/− , Rev7 −/− Rif1 −/− , Rev1 −/− , and Rev1 Δ9/Δ9 ; n = 3 for the other genotypes. In c , n = 7 for parental CH12F3 cells; n = 5 for Rev7 −/− ; n = 4 for 53bp1 −/− and Rev1 −/− ; n = 3 for the other genotypes. In d , n = 8 for parental CH12F3 cells; n = 4 for Rev7 −/− and Rev3l −/− ; n = 6 for Rev1 −/− ; n = 5 for Rev1 Δ9/Δ9 , n = 3 for the other genotypes. In e , n = 5 for parental CH12F3 cells; n = 3 for the other genotypes. In f , n = 8 for parental CH12F3 cells; n = 6 for Rev7 −/− ; n = 4 for Rev1 −/− , Rev1 Δ9/Δ9 , and Rev3l −/− ; n = 3 for 53bp1 −/− , Rif1 −/− , and Shld3 −/− . Three or more independent clones for each genotype were assayed and n represents independent experiments. Data are represented as mean ± SD; one-way ANOVA followed by Dunnett’s multiple comparisons test was performed for all panels. Data in knockouts are compared with those in parental CH12F3 cells for all panels. In d , an extra comparison is shown by using Rev7 −/− as the reference group to highlight the difference between DSBR deficiencies and TLS deficiencies. **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05, ns: p > 0.05. Source data are provided as a Source Data file.

Article Snippet: Antibodies for ATM (2873S; Cell Signaling; 1:1000), 53BP1 (NB100-304; NOVUSBIO; 1:1000), RIF1 (ab1213422; Abcam; 1:500), REV7 (A9861; Abclonal; 1:1000), REV1 (sc-393022; Santa Cruz; 1:1000), REV3L (GTX17515; Gene Tex; 1:1000), AID (A16217; Abclonal; 1:1000), MSH2 (ab227941; Abcam; 1:1000), β-actin (AC028; Abclonal; 1:10,000), FLAG (F1804, Sigma; 1:1000), β-Tubulin (A01030HRP; Abbkine; 1:10,000), glyceraldehyde 3-phosphate dehydrogenase (AB2000; Abways; 1:20,000), and Rabbit TrueBlot (18-8816-33; Ebioscience; 1:1000) were used in western blotting.

Techniques: Irradiation, Knock-Out, Clone Assay

Journal: Nature Communications

Article Title: REV7 is required for processing AID initiated DNA lesions in activated B cells

doi: 10.1038/s41467-020-16632-8

Figure Lengend Snippet: a Schematic illustration of critical residues on REV7 protein (left). Representative western blot of REV7 mutant proteins in B cells from three replicates are shown at right. EV: empty vector control. CSR levels to IgG1 ( b ), end resection level in Sγ1 region ( c ), and percentage of C > G transversion in 5′Sμ region ( d ) after LPS/IL4 stimulation at Day 4 of REV7-complementated primary B cells. CSR levels to IgA ( e ), cell numbers ( f ), and percentage of apoptotic population ( g ) of indicated CH12F3 cells with cytokine stimulation (CIT) at Day 3 are shown. n = 3 for each genotype in b – g and n represents independent experiments. Data are represented as mean ± SD, one-way ANOVA followed by Dunnett’s multiple comparisons test was performed for b – g . Data from cells complemented with WT REV7 protein are used as reference group in comparison. **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05, ns: p > 0.05. P -values and sample sizes are provided in Supplementary Table . Source data are provided as a Source Data file.

Article Snippet: Antibodies for ATM (2873S; Cell Signaling; 1:1000), 53BP1 (NB100-304; NOVUSBIO; 1:1000), RIF1 (ab1213422; Abcam; 1:500), REV7 (A9861; Abclonal; 1:1000), REV1 (sc-393022; Santa Cruz; 1:1000), REV3L (GTX17515; Gene Tex; 1:1000), AID (A16217; Abclonal; 1:1000), MSH2 (ab227941; Abcam; 1:1000), β-actin (AC028; Abclonal; 1:10,000), FLAG (F1804, Sigma; 1:1000), β-Tubulin (A01030HRP; Abbkine; 1:10,000), glyceraldehyde 3-phosphate dehydrogenase (AB2000; Abways; 1:20,000), and Rabbit TrueBlot (18-8816-33; Ebioscience; 1:1000) were used in western blotting.

Techniques: Western Blot, Mutagenesis, Plasmid Preparation

Journal: Nature Communications

Article Title: REV7 is required for processing AID initiated DNA lesions in activated B cells

doi: 10.1038/s41467-020-16632-8

Figure Lengend Snippet: a Assessment of Rev7 deletion efficiency. Schematic illustration of genotyping strategy of Rev7 -floxed allele (upper). Q-PCR primers are marks as arrows. A locus in Gapdh (chr6) was assayed as the loading control (blue arrows). Percentage of flowed allele are shown for Cd19creRev7 fl/fl B cells at Day 3 and 4 after stimulation (lower). Colored dot indicates data from same mouse. n = 5 for each genotype, n represents independent mice. Cell numbers ( b ) and percentage of apoptotic population ( c ) upon LPS/IL4 (upper) or LPS (lower) are plotted. d Genotyping of indicated CSR-activated B cells. e Percentage of cells in G2/M phase is plotted for the indicated genotypes. A −/− : Aicda −/− , A5 −/− : Aicda −/− 53bp1 −/− , ACR −/− : Aicda −/− Cd19creRev7 fl/fl . n = 3 for each genotype in b – e and n represents independent mice. Data are represented as mean ± SD. Two-tail paired t -test was performed for a and d , and one-way ANOVA followed by Dunnett’s multiple comparisons test was performed for b , c , and e . ACR −/− mice are used as reference group in comparison for b , c , and e . ** p < 0.01, * p < 0.05, ns: p > 0.05. P -values and sample sizes are provided in Supplementary Table . Source data are provided as a Source Data file.

Article Snippet: Antibodies for ATM (2873S; Cell Signaling; 1:1000), 53BP1 (NB100-304; NOVUSBIO; 1:1000), RIF1 (ab1213422; Abcam; 1:500), REV7 (A9861; Abclonal; 1:1000), REV1 (sc-393022; Santa Cruz; 1:1000), REV3L (GTX17515; Gene Tex; 1:1000), AID (A16217; Abclonal; 1:1000), MSH2 (ab227941; Abcam; 1:1000), β-actin (AC028; Abclonal; 1:10,000), FLAG (F1804, Sigma; 1:1000), β-Tubulin (A01030HRP; Abbkine; 1:10,000), glyceraldehyde 3-phosphate dehydrogenase (AB2000; Abways; 1:20,000), and Rabbit TrueBlot (18-8816-33; Ebioscience; 1:1000) were used in western blotting.

Techniques:

Journal: Nature Communications

Article Title: REV7 is required for processing AID initiated DNA lesions in activated B cells

doi: 10.1038/s41467-020-16632-8

Figure Lengend Snippet: Fractions of GC B cells (GL7 + Fas + ) from spleens ( a ) and Peyer’s patches ( b ) are showed with exampled flow cytometry plots and summary bar graphs. Each dot indicate result from an individual mouse. Six Cd19cre and seven Cd19creRev7 fl/fl mice were assayed for a and b . c Immunohistochemistry of spleens of indicated genotype. Representative images from three sets of mice are showed. Scale bar, 500 μm, is indicated as a black line on each picture. d Genotyping of indicated B (B220 + ) cell populations (PNA low : naive B cell, PNA high : GC B cell) in the spleen and Peyer’s patch. n = 5 mice. AID deficiency fully rescued the GC B-cell depletion in spleens ( e ) and Peyer’s patches ( f ) of Rev7-deficient mice. Four Cd19cre and five Cd19creRev7 fl/fl mice were assayed for e . Six Cd19cre and Cd19creRev7 fl/fl mice were assayed for f . Data are represented as mean ± SD. Two-tailed unpaired t -test was performed for a , b , e , and f ; two-tailed paired t -test was performed for d . ** p < 0.01, ns: p > 0.05. P -values and sample sizes are provided in Supplementary Table . Source data are provided as a Source Data file.

Article Snippet: Antibodies for ATM (2873S; Cell Signaling; 1:1000), 53BP1 (NB100-304; NOVUSBIO; 1:1000), RIF1 (ab1213422; Abcam; 1:500), REV7 (A9861; Abclonal; 1:1000), REV1 (sc-393022; Santa Cruz; 1:1000), REV3L (GTX17515; Gene Tex; 1:1000), AID (A16217; Abclonal; 1:1000), MSH2 (ab227941; Abcam; 1:1000), β-actin (AC028; Abclonal; 1:10,000), FLAG (F1804, Sigma; 1:1000), β-Tubulin (A01030HRP; Abbkine; 1:10,000), glyceraldehyde 3-phosphate dehydrogenase (AB2000; Abways; 1:20,000), and Rabbit TrueBlot (18-8816-33; Ebioscience; 1:1000) were used in western blotting.

Techniques: Flow Cytometry, Immunohistochemistry, Two Tailed Test

Journal: Nature Communications

Article Title: REV7 is required for processing AID initiated DNA lesions in activated B cells

doi: 10.1038/s41467-020-16632-8

Figure Lengend Snippet: Cell numbers ( a ) and percentage of apoptotic population ( b ) with(+)/without(−) cytokine stimulation (CIT) at Day 3 are showed for indicated genotypes. UM −/− : Ung −/− Msh2 −/− , UMR −/− : Ung −/− Msh2 −/− Rev7 −/− . Colored dots indicate individual clones. In a , n = 5 for parental CH12F3 cells; n = 6 for Rev7 −/− , Ung −/− Rev7 −/− (with CIT) and Msh2 −/− Rev7 −/− ; n = 3 for Ung −/− Rev7 −/− (without CIT); n = 4 for the other genotypes. In b , n = 6 for parental CH12F3 cells, Ung −/− Rev7 −/− and Msh2 −/− Rev7 −/− ; n = 5 for Rev7 −/− ; n = 4 for the other genotypes. Three independent clones for each genotype were assayed, and n represents independent experiments. Data are represented as mean ± SD. One-way ANOVA followed by Dunnett’s multiple comparisons test was performed for all panels. **** p < 0.0001, *** p < 0.001, ** p < 0.01, ns: p > 0.05. P -values and sample sizes are provided in Supplementary Table . Source data are provided as a Source Data file.

Article Snippet: Antibodies for ATM (2873S; Cell Signaling; 1:1000), 53BP1 (NB100-304; NOVUSBIO; 1:1000), RIF1 (ab1213422; Abcam; 1:500), REV7 (A9861; Abclonal; 1:1000), REV1 (sc-393022; Santa Cruz; 1:1000), REV3L (GTX17515; Gene Tex; 1:1000), AID (A16217; Abclonal; 1:1000), MSH2 (ab227941; Abcam; 1:1000), β-actin (AC028; Abclonal; 1:10,000), FLAG (F1804, Sigma; 1:1000), β-Tubulin (A01030HRP; Abbkine; 1:10,000), glyceraldehyde 3-phosphate dehydrogenase (AB2000; Abways; 1:20,000), and Rabbit TrueBlot (18-8816-33; Ebioscience; 1:1000) were used in western blotting.

Techniques: Clone Assay

Journal: PLOS Biology

Article Title: Canonical and noncanonical roles of Hop1 are crucial for meiotic prophase in the fungus Sordaria macrospora

doi: 10.1371/journal.pbio.3002705

Figure Lengend Snippet: ( A ) Schematic domain organization of Sordaria Hop1. ( B ) AlphaFold2 modelled Sordaria Hop1 HORMA domain (left), atomic (PDB:4TZJ), and experimentally determined C . elegans HIM-3 (middle) share structural similarities as shown by their superimposition (HIM-3 gray, right). Alpha-helices and beta-strands are in red and green, respectively. ( C ) Localization of Hop1 in WT meiosis. Hop1-GFP (left) and Spo76/Pds5-TdTomato (Tred, middle) are perfectly colocalized in the 2 pre-karyogamy haploid nuclei (top) and from leptotene to the post-pachytene diffuse stage (bottom). Scale bars: 2 μm. SCD, S/TJQ cluster domain; WT, wild-type.

Article Snippet: In addition to Hop1, as for most eukaryotes, the S . macrospora genome contains also 2 other HORMA domain proteins: the subunit of DNA polymerase Zeta Rev7 (SMAC_04698) and the spindle-checkpoint protein Mad2 (SMAC_09019) ( ).

Techniques:

Journal: PLOS Biology

Article Title: Canonical and noncanonical roles of Hop1 are crucial for meiotic prophase in the fungus Sordaria macrospora

doi: 10.1371/journal.pbio.3002705

Figure Lengend Snippet: ( A ) Top: domain diagram of WT Hop1. Bottom: diagram of the deleted or mutated Hop1 sites in the 6 analyzed mutants (). ( B ) Protein localization and phenotypes of the hop1-HORMAΔ mutant. Top: In the hop1Δ background, the mutant protein tagged with mCherry (mC, left) is not visible along the axes, marked by Spo76-GFP (middle) and merge (right). Bottom: colocalization with Ecm11-GFP + Hei10-GFP (middle) indicates that only few SC segments (containing Hei10 foci, arrows) are formed in this mutant; right corresponding DAPI. ( C, D ) hop1-HORMAΔ SCs exhibit the same length ( C ) and Hei10 foci number ( D ) as hop1Δ . Mean and error bar (SD) are indicated for each set. Significance between WT, hop1Δ and hop1-HORMAΔ nuclei was established by Brown–Forsythe ANOVA test: ns = not significant, P -value > 0.05; n = 38 and 42 nuclei. ( E ) hop1-only-HORMA . Top: the HORMA domain alone is sufficient for axis localization of the protein but only as discontinuous segments (arrows) that colocalize with Spo76-GFP (arrows middle and merge right) in contrast to the continuous lines seen in WT with Hop1-mCherry (right). Bottom: colocalization of Hop1-only-HORMA-mC (left) with Ecm11-GFP and Hei10-GFP (middle) and merge (right) indicates that the visible mutant segments correspond to SCs. ( C ) The mutant SCs have the same length as hop1Δ SCs ( C ) but exhibit a slightly lower number of Hei10 foci ( D ): Brown–Forsythe ANOVA test, ns = not significant; n = 31 and 42 nuclei. Scale bars: 2 μm. The raw data underlying panels 6C and 6D are available in . SC, synaptonemal complex; WT, wild-type.

Article Snippet: In addition to Hop1, as for most eukaryotes, the S . macrospora genome contains also 2 other HORMA domain proteins: the subunit of DNA polymerase Zeta Rev7 (SMAC_04698) and the spindle-checkpoint protein Mad2 (SMAC_09019) ( ).

Techniques: Mutagenesis