Journal: eLife

Article Title: A missense in HSF2BP causing primary ovarian insufficiency affects meiotic recombination by its novel interactor C19ORF57/BRME1

doi: 10.7554/eLife.56996

Figure Lengend Snippet:

Article Snippet: The primary antibodies used for immunofluorescence were rabbit αHSF2BP R2 (1:30, ProteintechTM), rabbit αBRME1 R2 (1:100, ProteintechTM), mouse αSYCP3 IgG sc-74569 (1:100, Santa Cruz), rabbit α-SYCP3 serum K921 (provided by Dr. José Luis Barbero, Centro de Investigaciones Biológicas, Spain), rabbit αSYCP1 IgG ab15090 (1:200, Abcam), rabbit anti-γH2AX (ser139) IgG #07–164 (1:500, Millipore), mouse αMLH1 51-1327GR (1:20, BD Biosciences), mouse αCDK2 (1:20; Santa Cruz Sc-6248) rabbit αRAD51 PC130 (1:50, Calbiochem), rabbit αRPA1 serum ¨Molly¨ (1:30, provided by Dr. Edyta Marcon, Medical Research University of Toronto, Canada), rat αRPA2 2208S (1:100, Cell Signaling), rabbit αDMC1 (1:500, ProteintechTM), rabbit αSPATA22 16989–1-AP (1:60, Proteintech), mouse αFlag IgG (1:100; F1804, Sigma-Aldrich).

Techniques: Recombinant, Generated, Sequencing, CRISPR, DNA Binding Assay, In Situ, cDNA Library Assay, Staining, Plasmid Preparation

Journal: eLife

Article Title: A missense in HSF2BP causing primary ovarian insufficiency affects meiotic recombination by its novel interactor C19ORF57/BRME1

doi: 10.7554/eLife.56996

Figure Lengend Snippet:

Article Snippet: The primary antibodies used for immunofluorescence were rabbit αHSF2BP R2 (1:30, ProteintechTM), rabbit αBRME1 R2 (1:100, ProteintechTM), mouse αSYCP3 IgG sc-74569 (1:100, Santa Cruz), rabbit α-SYCP3 serum K921 (provided by Dr. José Luis Barbero, Centro de Investigaciones Biológicas, Spain), rabbit αSYCP1 IgG ab15090 (1:200, Abcam), rabbit anti-γH2AX (ser139) IgG #07–164 (1:500, Millipore), mouse αMLH1 51-1327GR (1:20, BD Biosciences), mouse αCDK2 (1:20; Santa Cruz Sc-6248) rabbit αRAD51 PC130 (1:50, Calbiochem), rabbit αRPA1 serum ¨Molly¨ (1:30, provided by Dr. Edyta Marcon, Medical Research University of Toronto, Canada), rat αRPA2 2208S (1:100, Cell Signaling), rabbit αDMC1 (1:500, ProteintechTM), rabbit αSPATA22 16989–1-AP (1:60, Proteintech), mouse αFlag IgG (1:100; F1804, Sigma-Aldrich).

Techniques: Recombinant, Generated, Sequencing, CRISPR, DNA Binding Assay, In Situ, cDNA Library Assay, Staining, Plasmid Preparation

Journal: eLife

Article Title: A missense in HSF2BP causing primary ovarian insufficiency affects meiotic recombination by its novel interactor C19ORF57/BRME1

doi: 10.7554/eLife.56996

Figure Lengend Snippet: ( a, c ) Double labeling of γH2AX (green) and SYCP3 (red) in ( a ) spermatocyte and ( c ) oocyte spreads from WT, Hsf2bp S167L/S167L and Hsf2bp -/- mice. ( a ) Males display an accumulation of γH2AX patches in Hsf2bp S167L/S167L pachynemas and a strong accumulation in the whole nucleus in Hsf2bp -/- zygotene-like arrested cells. Plots on the right of the panel represent the percentage of pachynemas with γH2AX labeling (Nuclei: 364 Hsf2bp +/+ and 376 Hsf2bp S167L/S167L from three adult mice) and the quantification of γH2AX intensity on autosomes at early and mid-pachytene stages (Nuclei: 53 early and 60 mid pachynemas from three adult mice of each genotype). Two-tailed Welch’s t-test analysis: *p<0.05, ****p<0.0001. ( c ) In females there is an accumulation of γH2AX in Hsf2bp S167L/S167L pachynemas that is stronger in those from Hsf2bp -/- females. Nuclei: n = 21/20/19 pachynemas from 2 Hsf2bp +/+ / Hsf2bp S167L/S167L / Hsf2bp -/- embryos (17.5 dpc). Two-tailed Welch’s t-test analysis: *p<0.05, ****p<0.0001. ( b, d ) Double immunolabeling of RPA1 (green) and SYCP3 (red) in ( b ) spermatocyte and ( d ) oocyte spreads from Hsf2bp +/+ , Hsf2bp S167L/S167L and Hsf2bp -/- . ( b ) In males, RPA1 accumulates at early and mid-pachytene in S167L spermatocytes and in the zygotene-like arrested cells from Hsf2bp -/- . Plot on the right of the panel represents the quantification. Nuclei: n = 31/34/37 leptonemas, n = 29/29/37 zygonemas/zygonemas-like from three adult Hsf2bp +/+ , Hsf2bp S167L/S167L and Hsf2bp -/- mice respectively, n = 33 early and 46 mid pachynemas from three adult Hsf2bp +/+ and Hsf2bp S167L/S167L mice. Two-tailed Welch’s t-test analysis: ns, no significant differences; **p<0.01, ****p<0.0001. ( d ) In females, RPA1 labeling is similar in Hsf2bp +/+ , Hsf2bp S167L/S167L and Hsf2bp -/- oocytes at zygotene and pachytene. Plot on the right of the panel represents the quantification. Nuclei: Hsf2bp +/+ / Hsf2bp S167L/S167L / Hsf2bp -/- n = 42/41/23 zygonemas from two embryos (16.5 dpc) and n = 25/25/24 pachynemas from two embryos (17.5 dpc). Two-tailed Welch’s t-test analysis: ns, no significant differences. Bar in all panels, 10 μm. Extended panels for RPA1 figures in .

Article Snippet: The primary antibodies used for immunofluorescence were rabbit αHSF2BP R2 (1:30, ProteintechTM), rabbit αBRME1 R2 (1:100, ProteintechTM), mouse αSYCP3 IgG sc-74569 (1:100, Santa Cruz), rabbit α-SYCP3 serum K921 (provided by Dr. José Luis Barbero, Centro de Investigaciones Biológicas, Spain), rabbit αSYCP1 IgG ab15090 (1:200, Abcam), rabbit anti-γH2AX (ser139) IgG #07–164 (1:500, Millipore), mouse αMLH1 51-1327GR (1:20, BD Biosciences), mouse αCDK2 (1:20; Santa Cruz Sc-6248) rabbit αRAD51 PC130 (1:50, Calbiochem), rabbit αRPA1 serum ¨Molly¨ (1:30, provided by Dr. Edyta Marcon, Medical Research University of Toronto, Canada), rat αRPA2 2208S (1:100, Cell Signaling), rabbit αDMC1 (1:500, ProteintechTM), rabbit αSPATA22 16989–1-AP (1:60, Proteintech), mouse αFlag IgG (1:100; F1804, Sigma-Aldrich).

Techniques: Labeling, Two Tailed Test, Immunolabeling

Journal: eLife

Article Title: A missense in HSF2BP causing primary ovarian insufficiency affects meiotic recombination by its novel interactor C19ORF57/BRME1

doi: 10.7554/eLife.56996

Figure Lengend Snippet: ( a ) Double labeling of SPATA22 (green) and SYCP3 (red) in spermatocyte (upper panel) and oocyte (lower panel) spreads from WT, Hsf2bp -/- and Hsf2bp S167L/S167L mice. SPATA22 is accumulated in knock-out spermatocytes and oocytes and shows a milder accumulation in the Hsf2bp S167L/S167L spermatocytes. Hsf2bp S167L/S167L oocytes show a slight but not significant accumulation. Plots on the right of the panel represents the quantification of SPATA22 labeling. Males nuclei: n = 20 cells for each stage from two adult mice of each genotype. Females nuclei: Hsf2bp +/+ / Hsf2bp S167L/S167L /Hsf2bp -/- n = 41/40/40 zygonemas from two embryos and n = 40/39/38 pachynemas from two embryos (17.5 dpc). Two-tailed Welch’s t-test analysis: ns, no significant differences, **p<0.01, ****p<0.0001. ( b ) Double immunofluorescence of MLH1 (green) and SYCP3 (red) in spermatocyte spreads from WT, Hsf2bp S167L/S167L and Hsf2bp -/- . MLH1 foci are significantly reduced in the Hsf2bp S167L/S167L spermatocytes and absent in the knock-out. The plot on the right shows the quantification. See also for the plot showing the percentage of bivalents without CO. Nuclei: n = 61 for Hsf2bp +/+ , 89 for Hsf2bp S167L/S167L and 60 for Hsf2bp -/- from three adult mice of each genotype. Two-tailed Welch’s t-test analysis: ****p<0.0001. ( c ) Double labeling of CDK2 (green) and SYCP3 (red) in oocyte spreads from 17.5 dpc Hsf2bp +/+ , Hsf2bp S167L/S167L and Hsf2bp -/- embryos. During meiotic prophase I, CDK2 localizes to the telomeres of chromosomes from leptotene to diplotene. However, around mid-pachytene additional interstitial CDK2 signals appear at CO sites, colocalizing with MLH1. As a measure of COs, just interstitial CDK2 foci (non-telomeric) have been counted. Hsf2bp -/- and Hsf2bp S167L/S167L females show a high and moderate reduction in the number of COs, respectively. Plot on the right of the panel show the quantification. See also for the plot showing the percentage of bivalents without CO. Nuclei: Hsf2bp +/+ / Hsf2bp S167L/S167L /Hsf2bp -/- n = 79/67/46 from three embryos (17.5 dpc). Two-tailed Welch’s t-test analysis: ***p<0.0001, ****p<0.0001. ( d ) Double immunofluorescence of γH2AX (green) and SYCP3 (red) in spermatocyte spreads from WT and Hsf2bp S167L/S167L mice. At pachytene, γH2AX allows the identification of the XY bivalent. Diagram on the right represents the quantification of the pachynemas with unsynapsed sex chromosomes from Hsf2bp S167L/S167L and WT mice. Nuclei: n = 150 pachynemas from three adult mice of each genotype. Two-tailed Welch’s t-test analysis: *p<0.05. Bar in all panels, 10 μm.

Article Snippet: The primary antibodies used for immunofluorescence were rabbit αHSF2BP R2 (1:30, ProteintechTM), rabbit αBRME1 R2 (1:100, ProteintechTM), mouse αSYCP3 IgG sc-74569 (1:100, Santa Cruz), rabbit α-SYCP3 serum K921 (provided by Dr. José Luis Barbero, Centro de Investigaciones Biológicas, Spain), rabbit αSYCP1 IgG ab15090 (1:200, Abcam), rabbit anti-γH2AX (ser139) IgG #07–164 (1:500, Millipore), mouse αMLH1 51-1327GR (1:20, BD Biosciences), mouse αCDK2 (1:20; Santa Cruz Sc-6248) rabbit αRAD51 PC130 (1:50, Calbiochem), rabbit αRPA1 serum ¨Molly¨ (1:30, provided by Dr. Edyta Marcon, Medical Research University of Toronto, Canada), rat αRPA2 2208S (1:100, Cell Signaling), rabbit αDMC1 (1:500, ProteintechTM), rabbit αSPATA22 16989–1-AP (1:60, Proteintech), mouse αFlag IgG (1:100; F1804, Sigma-Aldrich).

Techniques: Labeling, Knock-Out, Two Tailed Test, Immunofluorescence

Journal: eLife

Article Title: A missense in HSF2BP causing primary ovarian insufficiency affects meiotic recombination by its novel interactor C19ORF57/BRME1

doi: 10.7554/eLife.56996

Figure Lengend Snippet: ( a, c ) Double labeling of γH2AX (green) and SYCP3 (red) in ( a ) spermatocyte and ( c ) oocyte spreads from WT and Brme1 -deficient mice. ( a ) In males γH2AX labeling appears in leptotene in both WT and Brme1 KO spermatocytes and increases during zygotene. However, whereas in the WT the signal decreases at pachytene, Brme1 -deficient mice show an accumulation of γH2AX in the zygotene-like arrest. The plot on the right represents the quantification of γH2AX intensity in both WT and KO spermatocytes. Nuclei: Brme1 +/+ / Brme1 -/- n = 20/20 leptotenes, 29/30 early zygonemas and 27/30 late zygonemas/zygonemas like from two adult mice. Two-tailed Welch's t-test analysis: ns, no significant differences; **p<0.01. ( c ) In females, γH2AX labeling accumulates at pachytene. Nuclei: Brme1 +/+ / Brme1 -/- n = 21/20 pachynemas from two embryos (17.5 dpc) of each genotype ( Brme1 +/+ from ). Two-tailed Welch's t-test analysis: ns, no significant differences; *p<0.05 ( b, d ) Double immunolabeling of RPA1 (green) and SYCP3 (red) in ( b ) spermatocyte and ( d ) oocyte spreads from Brme1 +/+ and Brme1 -/- mice. ( b ) In males, RPA1 accumulates at the late zygotene-like arrest of the Brme1 -/- mouse indicating a defective DNA repair in these mutants. Plot on the right of the panel represents the quantification of RPA1 foci on each genotype and stage. Nuclei: Brme1 +/+ / Brme1 -/- n = 31/26 leptonemas and 29/34 late zygonemas/zygonemas-like from three adult WT mice ( Brme1 +/+ from ) and 2 Brme1 -/- adult mice. Two-tailed Welch's t-test analysis: ns, no significant differences; ****p<0.0001. ( d ) In females, RPA1 foci are similar in Brme1 +/+ and Brme1 -/- oocytes at zygotene and pachytene. Nuclei: Brme1 +/+ / Brme1 -/- n = 42/22 zygonemas from two embryos (16.5 dpc) and 25/23 pachynemas from two embryos (17.5 dpc) of each genotype ( Brme1 +/+ from ). Two-tailed Welch'´s t-test analysis: ns, no significant differences. Bar in all panels, 10 μm.

Article Snippet: The primary antibodies used for immunofluorescence were rabbit αHSF2BP R2 (1:30, ProteintechTM), rabbit αBRME1 R2 (1:100, ProteintechTM), mouse αSYCP3 IgG sc-74569 (1:100, Santa Cruz), rabbit α-SYCP3 serum K921 (provided by Dr. José Luis Barbero, Centro de Investigaciones Biológicas, Spain), rabbit αSYCP1 IgG ab15090 (1:200, Abcam), rabbit anti-γH2AX (ser139) IgG #07–164 (1:500, Millipore), mouse αMLH1 51-1327GR (1:20, BD Biosciences), mouse αCDK2 (1:20; Santa Cruz Sc-6248) rabbit αRAD51 PC130 (1:50, Calbiochem), rabbit αRPA1 serum ¨Molly¨ (1:30, provided by Dr. Edyta Marcon, Medical Research University of Toronto, Canada), rat αRPA2 2208S (1:100, Cell Signaling), rabbit αDMC1 (1:500, ProteintechTM), rabbit αSPATA22 16989–1-AP (1:60, Proteintech), mouse αFlag IgG (1:100; F1804, Sigma-Aldrich).

Techniques: Labeling, Two Tailed Test, Immunolabeling

Journal: eLife

Article Title: A missense in HSF2BP causing primary ovarian insufficiency affects meiotic recombination by its novel interactor C19ORF57/BRME1

doi: 10.7554/eLife.56996

Figure Lengend Snippet:

Article Snippet: The primary antibodies used for immunofluorescence were rabbit αHSF2BP R2 (1:30, ProteintechTM), rabbit αBRME1 R2 (1:100, ProteintechTM), mouse αSYCP3 IgG sc-74569 (1:100, Santa Cruz), rabbit α-SYCP3 serum K921 (provided by Dr. José Luis Barbero, Centro de Investigaciones Biológicas, Spain), rabbit αSYCP1 IgG ab15090 (1:200, Abcam), rabbit anti-γH2AX (ser139) IgG #07–164 (1:500, Millipore), mouse αMLH1 51-1327GR (1:20, BD Biosciences), mouse αCDK2 (1:20; Santa Cruz Sc-6248) rabbit αRAD51 PC130 (1:50, Calbiochem), rabbit αRPA1 serum ¨Molly¨ (1:30, provided by Dr. Edyta Marcon, Medical Research University of Toronto, Canada), rat αRPA2 2208S (1:100, Cell Signaling), rabbit αDMC1 (1:500, ProteintechTM), rabbit αSPATA22 16989–1-AP (1:60, Proteintech), mouse αFlag IgG (1:100; F1804, Sigma-Aldrich).

Techniques: Recombinant, Generated, Sequencing, CRISPR, DNA Binding Assay, In Situ, cDNA Library Assay, Staining, Plasmid Preparation