Journal: PLoS ONE

Article Title: Fidgetin-Like1 Is a Strong Candidate for a Dynamic Impairment of Male Meiosis Leading to Reduced Testis Weight in Mice

doi: 10.1371/journal.pone.0027582

Figure Lengend Snippet: List of genes and annoted sequences localized between 9.6 and 14.7 Mbp on Mouse Chromosome 11.

Article Snippet: Testis sections were deparaffinized, rehydrated and then treated either by 0.2% TX-100 in PBS for 5 minutes or by 3×5 minutes microwave in citrate buffer (0.1 M sodium citrate and 0.1 M citric acid, pH 6.0) just before blockade of peroxidase activity by incubation with 3% H 2 O 2 for 10 min. After non specific binding site saturation, sections were incubated with anti FIGNL1 antibody (Novus Biologicals) (1/100 or 1/50 in blocking solution) for one hour at room temperature and then with horseradish peroxidase-conjugated anti-rabbit antibody.

Techniques: Binding Assay

Journal: PLoS ONE

Article Title: Fidgetin-Like1 Is a Strong Candidate for a Dynamic Impairment of Male Meiosis Leading to Reduced Testis Weight in Mice

doi: 10.1371/journal.pone.0027582

Figure Lengend Snippet: a ) Polymorphism map of spretus fignl1 gene. By PCR and sequencing of spretus Fignl1, we could observe 9 coding variants compared to the B6 version. These variants are mainly located (8 of them) in the N terminal region of Fignl1, reported as less conserved. In red are represented variants that could give rise to strong and significant variations in the protein structure or to post-translational modifications. S99C may generate disulphide bonds with other cystein residues, H379Y generates a phosphorylable tyrosine, and M397K is specifically encountered in Mus spretus, by contrast with all other species, including monotremes, birds, amphibians and fish . b ) RT-PCR amplification of fignl1 ORF on testis cDNA of B6, SEG and 97C males, with primers spanning the complete ORF. The specific amplification product observed when a spretus allele is present corresponds to a mRNA smaller of about 1400 bp which may generate a truncated isoform of the protein; It is interesting to notice, that this isoform should not be problematic in spretus since it is normally present in this species.

Article Snippet: Testis sections were deparaffinized, rehydrated and then treated either by 0.2% TX-100 in PBS for 5 minutes or by 3×5 minutes microwave in citrate buffer (0.1 M sodium citrate and 0.1 M citric acid, pH 6.0) just before blockade of peroxidase activity by incubation with 3% H 2 O 2 for 10 min. After non specific binding site saturation, sections were incubated with anti FIGNL1 antibody (Novus Biologicals) (1/100 or 1/50 in blocking solution) for one hour at room temperature and then with horseradish peroxidase-conjugated anti-rabbit antibody.

Techniques: Sequencing, Reverse Transcription Polymerase Chain Reaction, Amplification

Journal: PLoS ONE

Article Title: Fidgetin-Like1 Is a Strong Candidate for a Dynamic Impairment of Male Meiosis Leading to Reduced Testis Weight in Mice

doi: 10.1371/journal.pone.0027582

Figure Lengend Snippet: From a cell type point of view, the location of the protein does not appear different between 97 C, B6 and SEG. (a and d) correspond to images from 97 C testes, (b and e) images from B6 testes and (c and f) images from SEG testes taken at objective ×100. (a, b and c) show stages VI–VIII of the spermatogenesis, close to spermiation. The Fignl1 is concentrated in the cytoplasm of mid-pachytene spermatocytes in a crescent-like pattern (see thin arrows in the enlarged views of the dashed area in a, b and c). To note the absence of staining in the spermatids (large arrows in the enlarged views). (d, e and f) show a stage XII with immunoreactive spermatocytes in metaphase. Notice in the enlargements presented at the right side of d, e and f pictures, the very particular location of Fignl1 around the achromatic spindle. This observation is representative of metaphases in B6 mice. In 97 C, a diffuse location of Fignl1 around the chromosomes could also be observed.

Article Snippet: Testis sections were deparaffinized, rehydrated and then treated either by 0.2% TX-100 in PBS for 5 minutes or by 3×5 minutes microwave in citrate buffer (0.1 M sodium citrate and 0.1 M citric acid, pH 6.0) just before blockade of peroxidase activity by incubation with 3% H 2 O 2 for 10 min. After non specific binding site saturation, sections were incubated with anti FIGNL1 antibody (Novus Biologicals) (1/100 or 1/50 in blocking solution) for one hour at room temperature and then with horseradish peroxidase-conjugated anti-rabbit antibody.

Techniques: Staining

Journal: PLoS ONE

Article Title: Fidgetin-Like1 Is a Strong Candidate for a Dynamic Impairment of Male Meiosis Leading to Reduced Testis Weight in Mice

doi: 10.1371/journal.pone.0027582

Figure Lengend Snippet: (a) Western blot revealed by anti-FIGNL1 antibody on extracts of B6, 97 C and SEG testes (50 µg of protein extract per lane). β-actin is taken as an internal control of loading. The Fignl1 band migrates around 74 kDa. There is no obvious difference in size and quantity between the two mouse species, as well as in the 97 C IRCS. The alternate splicing should generate a ∼6 kDa peptide that was not visible in our electrophoresis conditions. (b) Western blot revelation of the Fignl1 during the first wave of the spermatogenesis in B6 testis from 10 to 32 days post partum (DPP). Fignl1 is not detectable at 10 DPP, date of the meiosis entry but is observed at 16 DPP, and afterward, when spermatocytes appear in the tubules.). β-actin is taken as an internal control of loading. To note: the 7 kD isoform described in the text could not be detected in this 12% polyacrylamide gel. (c) Two-dimensional Western blot of protein extracts of B6 and 97 C testes revealed by anti FIGNL1. Fignl1 migrates as two principal spots at 76 kDa and 72 kDa for a same 5.86 pI. The 2D migration profile was the same for B6 and 97 C.

Article Snippet: Testis sections were deparaffinized, rehydrated and then treated either by 0.2% TX-100 in PBS for 5 minutes or by 3×5 minutes microwave in citrate buffer (0.1 M sodium citrate and 0.1 M citric acid, pH 6.0) just before blockade of peroxidase activity by incubation with 3% H 2 O 2 for 10 min. After non specific binding site saturation, sections were incubated with anti FIGNL1 antibody (Novus Biologicals) (1/100 or 1/50 in blocking solution) for one hour at room temperature and then with horseradish peroxidase-conjugated anti-rabbit antibody.

Techniques: Western Blot, Control, Electrophoresis, Migration

Journal: Nature communications

Article Title: FLIP(C1orf112)-FIGNL1 complex regulates RAD51 chromatin association to promote viability after replication stress.

doi: 10.1038/s41467-024-45139-9

Figure Lengend Snippet: Fig. 4 | FLIP forms a complex with FIGNL1. a 293 T cells were transfected with empty vector (EV), FLAG-FIGNL1 or GFP-FLIP as indicated for 48 h. Blots show association of FIGNL1 and FLIP, n = 3 independent experiments. b 293 T cells were transfected with EV or FLAG-FLIP for 48 h. Western blots show pull down of endogenous FIGNL1 following coIPs, n = 3 independent experiments. c 293 T cells were transfected with EV, FLAG-FIGNL1 or GFP-FLIP as indicated for 48 h. Cells were then treated with 2 μM cisplatin for 24 h prior to coIP, n = 2 independent experi- ments. d 293 T cells were transfected with FIGNL1-GFP together with either full-

Article Snippet: The antibodies used in this work are as follows: Anti-HA 1:1000 (Sigma, H3663-200), c1orf112 1:1000 (Sigma, HPA023778), FANCD2 F17 1:200 (Santa Cruz Biotechnology, sc-20022), FANCA 1:1000 (Bethyl, A301980A), tubulin 1:1000 (Sigma, T4026-.2ML), FANCI 1:1000 (Bethyl, A301-254A), Vinculin 1:1000 (Sigma, V9131-.2ML), ATM 1:1000 (Abcam, ab81292), γH2AX (JBW301) 1:1000 (Sigma, 05-636),mouseM2 anti-FLAG 1:1000 (Sigma, F1804-200UG), Rabbit anti-GFP antibody 1:1000 (Abcam, ab6556), RPA32-P-S4/8 1:1000 (Bethyl, A300-245A), RPA32 9H8 1:200 (Santa Cruz Biotechnology, sc-56770), CHK1-P-S317 1:1000 (Cell Signaling, 2344S), CHK1 G4 1:200 (Santa Cruz Biotechnology, sc-8408), FIGNL1 1:1000 (Proteintech, 17604-1-AP-150UL), GAPDH 1:8000 (Santa Cruz Biotechnology, sc-47724), ORC2 1:1000 (Abcam, ab68348), RAD51 1:1000 (Abcam, ab63801), Mouse monoclonal RAD51 1:1000 (Millipore-Sigma, 05-530-I), Rad51 Antibody (H92) 1:1000 (Santa Cruz Biotechnology, sc-8349), Goat anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody Alexa Fluor Plus 488 1:400 (Invitrogen, A32731), Goat anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody Alexa Fluor Plus 488 1:400 (Invitrogen, A32723), Goat anti-Rat IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 1:400 (Invitrogen, A48262), Goat anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody Alexa Fluor Plus 594 1:400 (Invitrogen, A32742), Goat anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody Alexa Fluor Plus 594 1:400 (Invitrogen, A32740).

Techniques: Transfection, Plasmid Preparation, Western Blot

Journal: Nucleic Acids Research

Article Title: The human Shu complex functions with PDS5B and SPIDR to promote homologous recombination

doi: 10.1093/nar/gkz738

Figure Lengend Snippet: BioID identifies SPIDR and PDS5B as novel binding partners of the Shu complex. ( A ) RPE-1 cells were transiently transfected with either HA-SWS1 or myc-SWSAP1 alone or with Flag-SPIDR. Flag-SPIDR was immunoprecipitated using anti-Flag-conjugated beads (Flag IP) and western blotted for SWS1 or SWSAP1 (co-IP) using anti-HA or anti-myc, antibodies, respectively. Inputs represent 10% of the protein lysate. ( B ) RPE-1 cells were transiently transfected with either HA-SWS1 or myc-SWSAP1 alone or with Flag-PDS5B N-terminal fragment (1–529 amino acids). Flag-PDS5B was immunoprecipitated using anti-Flag-conjugated beads (Flag IP) and western blotted for SWS1 or SWSAP1 (co-IP) using anti-HA or anti-myc antibodies, respectively. Inputs represent 10% of the protein lysate. ( C ) The PJ694a yeast strain was transformed with three plasmids; 1) a plasmid where SPIDR was fused to the GAL4-DNA binding domain (BD; pGBD-SPIDR), 2) a plasmid where either SWSAP1 or SWS1 was fused to the GAL4-DNA activating domain (AD; pGAD-SWSAP1 or pGAD-SWS1), and 3) a plasmid that constitutively expressed either SWS1 or SWSAP1 (pRS416-SWS1, pRS416-SWSAP1). A yeast-three-hybrid interaction between SPIDR and SWSAP1 or SWS1 was assayed by platting the yeast on SC-LEU-TRP-URA-HIS (Interaction, indicated by growth) and compared to the loading control SC-LEU-TRP-URA (Control). Empty BD (pGBD), AD (pGAD), and pRS416 plasmids were used as negative controls. ( D ) The PJ69a yeast strain was transformed with a plasmid where FIGNL1 was fused to the GAL4-DNA activating domain (AD; pGAD-FIGNL1) and 2) a plasmid where SWSAP1, SWS1, or SPIDR was fused to the GAL4-DNA binding domain (BD; pGBD-SWSAP1, pGBD-SWS1, pGBD-SPIDR). For the yeast-three-hybrids, a third plasmid that constitutively expressed either SWS1 or SWSAP1 (pRS416-SWS1, pRS416-SWSAP1) was also co-transformed. A yeast-two-hybrid interaction between FIGNL1 with SWS1, SWSAP1, or SPIDR was assayed by plating the yeast on SC-LEU-TRP-HIS (Interaction, indicated by growth) and compared to the loading control SC-LEU-TRP (Control). A yeast-three-hybrid interaction between SPIDR and SWSAP1 or SWS1 was assayed by plating the yeast on SC-LEU-TRP-URA-HIS (Interaction, indicated by growth) and compared to the loading control SC-LEU-TRP-URA (Control). Empty BD (pGBD), AD (pGAD), and pRS416 plasmids were used as negative controls.

Article Snippet: The FIGNL1 cDNA was purchased from Sino Biologicals (HG15206-G).

Techniques: Binding Assay, Transfection, Immunoprecipitation, Western Blot, Co-Immunoprecipitation Assay, Transformation Assay, Plasmid Preparation

Journal: International Journal of Molecular Sciences

Article Title: 3-Methyl-4-Nitrophenol Exposure Deteriorates Oocyte Maturation by Inducing Spindle Instability and Mitochondrial Dysfunction

doi: 10.3390/ijms25073572

Figure Lengend Snippet: Figure 3. PNMC exposure disturbs spindle localization of Fignl1 in oocytes. (A–C) The mRNA levels of KATNAL1, FIGNL1, and SPAST were confirmed by qRT-PCR. The mRNA expressions of SPAST and

Article Snippet: Rabbit anti-Fignl1 polyclonal antibody (Cat# NBP2-47456) was procured from Novus Biologicals (Centennial, CO, USA); mouse anti-α-tubulin-FITC antibody (Cat# F2168) was purchased from Sigma Chemical Company (St. Louis, MO, USA); DyLight 549- conjugated goat anti-rabbit IgG (H + L) from Abbkine Biotechnology (San Diego, CA, USA), and IBMX (Cat# HY-12318) was obtained from MedChemExpress Company (Monmouth Junction, NJ, USA).

Techniques: Quantitative RT-PCR

Journal: Nature Communications

Article Title: FIGNL1 AAA+ ATPase remodels RAD51 and DMC1 filaments in pre-meiotic DNA replication and meiotic recombination

doi: 10.1038/s41467-023-42576-w

Figure Lengend Snippet: a Scheme of mouse Fignl1 flox and Δ alleles. Yellow and blue boxes and black triangles represent untranslated regions (UTRs), coding sequence (CDS), and loxP sequences, respectively. b Representative image of testes from mice with indicated genotypes. c Testis weights of adult mice (9–20 weeks) with indicated genotypes. The red bars are means. d Immunoblotting of FIGNL1 from whole-testis extracts of mice with indicated genotypes. α-tubulin is a loading control. e Sperm numbers of mice with indicated genotypes. The red bars are means. f Representative images of periodic acid-Schiff (PAS)-stained seminiferous tubule sections from the testis of Fignl1 +/+ Stra8-Cre + (Ctrl) and Fignl1 flox/Δ Stra8-Cre + ( Fignl1 cKO) mice. Sg, spermatogonia; Sc (L), leptotene spermatocytes; Sc (P), pachytene spermatocytes; St, spermatids. g Distribution of different meiotic prophase I stages in Ctrl (gray bars) and Fignl1 cKO (blue open bars). >100 cells were counted from each animal. The bar graphs indicate means. The red bars are SDs from three animals of each genotype. h Distribution of FIGNL1-positive and -negative spermatocytes in Ctrl (gray bars) and Fignl1 cKO (blue open bars). >100 leptotene/zygotene (lept./zyg.) and pachytene/diplotene (pach./dipl.) cells were analyzed from each animal. The means of two mice of each genotype are indicated. Representative images of testicular cell squashes are shown in Supplementary Fig. . i Representative images of spermatocyte-chromosome spreads immunostained for SYCP1 (green) and SYCP3 (magenta) at zygonema in Ctrl and Fignl1 cKO. The bottom panels are magnified images of regions with yellow dotted rectangles. j Quantification of the frequency of synapsis defects in Ctrl (gray bars) and Fignl1 cKO (blue open bars). >90 cells were analyzed from each animal. The bar graphs indicate means. The red bars are SDs from three mice of each genotype. In ( f )–( i ), genotypes of indicated animals are Ctrl, Fignl1 +/+ Stra8-Cre + ; Fignl1 cKO, Fignl1 flox/Δ Stra8-Cre + . The results of two-tailed unpaired t -tests are indicated in the graphs: **** p ≤ 0.0001. Scale bars, 50 μm in ( f ) and 10 μm for whole-nucleus images and 2 μm for magnified images in ( i ). Source data are provided as a Source Data file.

Article Snippet: This is consistent with the reported lethality of Fignl1 knockout (KO) mice in the International Mouse Phenotyping Consortium (IMPC) ( https://www.mousephenotype.org ).

Techniques: Sequencing, Western Blot, Control, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: FIGNL1 AAA+ ATPase remodels RAD51 and DMC1 filaments in pre-meiotic DNA replication and meiotic recombination

doi: 10.1038/s41467-023-42576-w

Figure Lengend Snippet: a Representative images of spermatocyte-chromosome spreads immunostained for RPA2 (white in the top panels and green in the bottom panels) and SYCP3 (magenta) at indicated meiotic prophase I stages in Ctrl and Fignl1 cKO. b Quantification of RPA2 focus numbers at different meiotic prophase I stages in Ctrl (black circles) and Fignl1 cKO (blue open circles). The red bars are means. c Representative images of spermatocyte-chromosome spreads immunostained for MSH4 (white in the left panels and green in the right panels), SYCP1 (magenta) and SYCP3 (blue) at midzygonema in Ctrl and Fignl1 cKO. The bottom panels are magnified images of regions with dotted yellow. d Quantification of MSH4 focus numbers at mid-zygonema in Ctrl (black circles) and Fignl1 cKO (blue open circles). The red bars are means. e The correlation between MSH4 focus number and total SC length at midzygonema in Ctrl (black circles) and Fignl1 cKO (blue open circles). Pearson’s r is shown in parentheses. A black solid line and a blue dashed line indicate linear regression for Ctrl and Fignl1 cKO, respectively. f Quantification of MSH4 focus density on the SCs at midzygonema in Ctrl (black circles) and Fignl1 cKO (blue open circles). The red bars are means. In ( e ) and ( f ), midzygotene nuclei with 40–120 μm of total SC length per nucleus were analyzed. Genotypes of indicated animals are: Ctrl, Fignl1 +/+ Stra8-Cre + ; Fignl1 cKO, Fignl1 flox/Δ Stra8-Cre + . The results of the two-tailed Mann–Whitney U -test are indicated in the graphs: **** p ≤ 0.0001. The total number of cells analyzed is indicated below the graphs. Scale bars in ( a ) and ( c ), 10 μm and 2 μm for magnified images in ( c ). Source data are provided as a Source Data file.

Article Snippet: This is consistent with the reported lethality of Fignl1 knockout (KO) mice in the International Mouse Phenotyping Consortium (IMPC) ( https://www.mousephenotype.org ).

Techniques: Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: FIGNL1 AAA+ ATPase remodels RAD51 and DMC1 filaments in pre-meiotic DNA replication and meiotic recombination

doi: 10.1038/s41467-023-42576-w

Figure Lengend Snippet: a Representative images of spermatocyte-chromosome spreads immunostained for DMC1 (white in the top panels and green in the bottom panels) and SYCP3 (magenta) at indicated meiotic prophase I stages in Ctrl and Fignl1 cKO. b Quantification of DMC1 focus numbers at different meiotic prophase I stages in Ctrl (black circles) and Fignl1 cKO (blue open circles). The red bars are means. c Representative images of spermatocyte-chromosome spreads immunostained for RAD51 (white in the top panels and green in the bottom panels) and SYCP3 (magenta) at indicated meiotic prophase I stages in Ctrl and Fignl1 cKO. d Quantification of RAD51 focus numbers at different meiotic prophase I stages in Ctrl (black circles) and Fignl1 cKO (blue open circles). The red bars are means. e Quantification of focus numbers of RAD51 (green), DMC1 (magenta), and RAD51-DMC1 colocalization (brown) at different meiotic prophase I stages in Ctrl (filled circles) and Fignl1 cKO (open circles). The black bars are means. Representative images of spermatocyte-chromosome spreads are shown in Supplementary Fig. . f , g Quantification of the frequency of RAD51-DMC1 colocalization at different meiotic prophase I stages in Ctrl (filled circles) and Fignl1 cKO (open circles). The degrees of RAD51 foci colocalizing with DMC1 ( f , green) and DMC1 foci colocalizing with RAD51 ( g , magenta) are shown. The black bars are means. Genotypes of indicated animals are: Ctrl, Fignl1 +/+ Stra8-Cre + ; Fignl1 cKO, Fignl1 flox/Δ Stra8-Cre + . The results of the two-tailed Mann–Whitney U -test are indicated in the graphs: **** p ≤ 0.0001. The total number of cells analyzed is indicated below the graphs. Scale bars in ( a ) and ( c ), 10 μm. Prelept., preleptonema; lept., leptonema; zyg., zygonema. Source data are provided as a Source Data file.

Article Snippet: This is consistent with the reported lethality of Fignl1 knockout (KO) mice in the International Mouse Phenotyping Consortium (IMPC) ( https://www.mousephenotype.org ).

Techniques: Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: FIGNL1 AAA+ ATPase remodels RAD51 and DMC1 filaments in pre-meiotic DNA replication and meiotic recombination

doi: 10.1038/s41467-023-42576-w

Figure Lengend Snippet: a Representative images of spermatocyte-chromosome spreads immunostained for RAD51 (left), SYCP3 (middle) and EdU (right, 60’ labeling) at preleptonema in Ctrl and Fignl1 cKO. b Quantification of RAD51 focus numbers at preleptonema in Ctrl (black circles) and Fignl1 cKO (blue open circles). The red solid bars and dashed bars are means for total cells and RAD51-positive cells (with >10 RAD51 foci; 26/38 and 25/48 cells in early and mid/late preleptonema, respectively), respectively. c Representative images of spermatocyte-chromosome spreads immunostained for DMC1 (left), SYCP3 (middle), and EdU (right, 60’ labeling) at preleptonema in Ctrl and Fignl1 cKO. d Quantification of DMC1 focus numbers at preleptonema in Ctrl (black circles) and Fignl1 cKO (blue open circles). The red solid bars and dashed bars are means for total cells and DMC1-positive cells (with >30 DMC1 foci; 28/39 and 26/45 cells in early and mid/late preleptonema, respectively), respectively. Genotypes of indicated animals are: Ctrl, Fignl1 +/+ Stra8-Cre + ; Fignl1 cKO, Fignl1 flox/Δ Stra8-Cre + . The results of the two-tailed Mann-Whitney U -test are indicated in the graphs: **** p ≤ 0.0001. The total number of cells analyzed is indicated below the graphs. Scale bars in ( a ) and ( c ), 10 μm. Source data are provided as a Source Data file.

Article Snippet: This is consistent with the reported lethality of Fignl1 knockout (KO) mice in the International Mouse Phenotyping Consortium (IMPC) ( https://www.mousephenotype.org ).

Techniques: Labeling, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: FIGNL1 AAA+ ATPase remodels RAD51 and DMC1 filaments in pre-meiotic DNA replication and meiotic recombination

doi: 10.1038/s41467-023-42576-w

Figure Lengend Snippet: a , c Representative images of spermatocyte-chromosome spreads immunostained for RAD51 ( a ), DMC1 ( c ) (white in the top panels and green in the bottom panels) and SYCP3 (magenta) at leptonema in Spo11 KO, Fignl1 cKO and Spo11 KO Fignl1 cKO. b , d Quantification of RAD51 ( b ) and DMC1 ( d ) focus numbers at different meiotic prophase I stages in Spo11 KO (orange circles), Fignl1 cKO (blue open circles), and Spo11 KO Fignl1 cKO (purple open circles). The red bars are means. Cells in which Stra8-Cre recombinase seemed to fail excision of the Fignl1 Δ allele in Spo11 KO Fignl1 cKO (<30 RAD51 foci or <60 DMC1 foci) are excluded. Quantification including these cells is shown in Supplementary Fig. . e Representative images of spermatocyte-chromosome spreads immunostained for RAD51 (green), DMC1 (magenta), and SYCP3 (blue) at late zygonema/early leptonema in Spo11 KO Fignl1 cKO. The bottom panels are magnified images of regions with yellow dotted rectangles. f Quantification of focus numbers of RAD51 (green open circles), DMC1 (magenta open circles), and RAD51-DMC1 colocalization (brawn open circles) at different meiotic prophase I stages in Spo11 KO Fignl1 cKO. The black bars are means. g , h Quantification of the frequency of RAD51-DMC1 colocalization at different meiotic prophase I stages in Spo11 KO Fignl1 cKO. The degrees of RAD51 foci colocalizing with DMC1 ( g , green open circles) and DMC1 foci colocalizing with RAD51 ( h , magenta open circles) are shown. The black bars are means. Genotypes of indicated animals are: Spo11 KO, Spo11 −/− Fignl1 flox/Δ ; Fignl1 cKO, Fignl1 flox/Δ Stra8-Cre + ; Spo11 KO Fignl1 cKO, Spo11 −/− Fignl1 flox/Δ Stra8-Cre + . The results of the two-tailed Mann–Whitney U -test are indicated in the graphs: **** p ≤ 0.0001. The total number of cells analyzed is indicated below the graphs. Scale bars in ( a ), ( c ) and ( e ), 10 μm and 2 μm for magnified images in ( e ). Prelept., preleptonema; lept., leptonema; zyg., zygonema. Source data are provided as a Source Data file.

Article Snippet: This is consistent with the reported lethality of Fignl1 knockout (KO) mice in the International Mouse Phenotyping Consortium (IMPC) ( https://www.mousephenotype.org ).

Techniques: Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: FIGNL1 AAA+ ATPase remodels RAD51 and DMC1 filaments in pre-meiotic DNA replication and meiotic recombination

doi: 10.1038/s41467-023-42576-w

Figure Lengend Snippet: a RAD51 ChIP-SSDS signals (green) in Ctrl and Fignl1 cKO with Spo11-oligo counts (blue) and DMC1 ChIP-SSDS signals (black) in wild-type at an indicated chromosomal region with a hotspot on mouse chromosome 2. b The heatmap of RAD51 ChIP-SSDS signals (green) in Ctrl and Fignl1 cKO and Spo11-oligo counts (blue) and DMC1 ChIP-SSDS signals (black) in wild-type. The 4000 most active meiotic recombination hotspots on autosomes identified by Spo11-oligo in wild-type are ordered by SPO11-oligos counts . ChIP-SSDS signals are centered relative to hotspot centers. In ( a ) and ( b ), Spo11-oligo and DMC1 ChIP-SSDS data are from previous studies , . c Representative images of spermatocyte-chromosome spreads immunostained for RAD51 (green), RPA2 (magenta), and SYCP3 (blue) at midzygonema in Ctrl and Fignl1 cKO. The bottom panels are magnified images of regions with yellow dotted rectangles. Scale bars, 10 μm for whole-nucleus images and 2 μm for magnified images. d Quantification of focus numbers of RAD51 (green), RPA2 (magenta) and RAD51-RPA2 colocalization (brawn) at mid zygonema in Ctrl (filled circles) and Fignl1 cKO (open circles). The black bars are means. e Quantification of the frequency of RAD51-RPA2 colocalization at midzygonema in Ctrl (black circles) and Fignl1 cKO (blue open circles). The degree of RPA2 foci colocalizing with RAD51 is shown. The red bars are means. Genotypes of indicated animals are: Ctrl, Fignl1 +/+ Stra8-Cre + ; Fignl1 cKO, Fignl1 flox/Δ Stra8-Cre + . The results of the two-tailed Mann–Whitney U -test are indicated: **** p ≤ 0.0001. The total number of cells analyzed is indicated below the graphs. Source data are provided as a Source Data file.

Article Snippet: This is consistent with the reported lethality of Fignl1 knockout (KO) mice in the International Mouse Phenotyping Consortium (IMPC) ( https://www.mousephenotype.org ).

Techniques: Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: FIGNL1 AAA+ ATPase remodels RAD51 and DMC1 filaments in pre-meiotic DNA replication and meiotic recombination

doi: 10.1038/s41467-023-42576-w

Figure Lengend Snippet: a Representative images of spermatocyte-chromosome spreads immunostained for RAD51 (white in the top panels and green in the bottom panels) and SYCP3 (magenta) at early zygonema in Ctrl, Swsap1 KO , Fignl1 cKO and Swsap1 KO Fignl1 cKO. b Quantification of RAD51 focus numbers at different meiotic prophase I stages in Ctrl (black circles), Swsap1 KO (orange circles), Fignl1 cKO (blue open circles), and Swsap1 KO Fignl1 cKO (purple open circles). The red bars are means. c Representative images of spermatocyte-chromosome spreads immunostained for DMC1 (white in the top panels and green in the bottom panels) and SYCP3 (magenta) at early zygonema in Ctrl, Swsap1 KO , Fignl1 cKO, and Swsap1 KO Fignl1 cKO. d Quantification of DMC1 focus numbers at different meiotic prophase I stages in Ctrl (black circles), Swsap1 KO (orange circles), Fignl1 cKO (blue open circles), and Swsap1 KO Fignl1 cKO (purple open circles). The red bars are means. Genotypes of indicated animals are: Swsap1 KO, Swsap1 −/− Fignl1 flox/Δ ; Fignl1 cKO, Fignl1 flox/Δ Stra8-Cre + ; Swsap1 KO Fignl1 cKO, Swsap1 −/− Fignl1 flox/Δ Stra8-Cre + . The results of the two-tailed Mann-Whitney U -test are indicated in the graphs: **** p ≤ 0.0001. The total number of cells analyzed is indicated below the graphs. Scale bars in ( a ) and ( c ), 10 μm. Source data are provided as a Source Data file.

Article Snippet: This is consistent with the reported lethality of Fignl1 knockout (KO) mice in the International Mouse Phenotyping Consortium (IMPC) ( https://www.mousephenotype.org ).

Techniques: Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: FIGNL1 AAA+ ATPase remodels RAD51 and DMC1 filaments in pre-meiotic DNA replication and meiotic recombination

doi: 10.1038/s41467-023-42576-w

Figure Lengend Snippet: a Electrophoresis mobility shift assay showing in vitro disruption of RAD51-DNA complexes by FIGNL1. A representative gel image among triplicate is shown. Human RAD51 protein was incubated with either Cy5-labeled ssDNA (40nt) or dsDNA (40 bp) in the presence of ATP. After the formation of the protein-DNA complex, purified human FIGNL1ΔN was added at the indicated concentration and further incubated. The complexes were analyzed by polyacrylamide gel electrophoresis and the fluorescence from the DNAs was captured. b Quantification of free ssDNA or dsDNA in ( a ). The red bars are means and SDs from three independent experiments. c Model of FIGNL1 function in spermatocytes. In pre-meiotic S-phase and early meiotic prophase I, FIGNL1 dissociates RAD51 and DMC1 from dsDNAs. At meiotic DSB sites, FIGNL1 removes RAD51 and DMC1 from ssDNAs, which facilitates efficient loading of MutSγ (MSH4-MSH5) and further processing of homologous recombination to crossover outcome. Source data are provided as a Source Data file.

Article Snippet: This is consistent with the reported lethality of Fignl1 knockout (KO) mice in the International Mouse Phenotyping Consortium (IMPC) ( https://www.mousephenotype.org ).

Techniques: Electrophoresis, Mobility Shift, In Vitro, Disruption, Incubation, Labeling, Purification, Concentration Assay, Polyacrylamide Gel Electrophoresis, Fluorescence, Homologous Recombination