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: 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