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hormad1 rabbit polyclonal antibody (Novus Biologicals)

Name: hormad1 rabbit polyclonal antibody
Brand: Novus Biologicals
Rating: 93 (3 reviews)

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Novus Biologicals hormad1 rabbit polyclonal antibody

Figure 1. Signiﬁcant increase in the ectopic expression of <t>HORMAD1</t> in Squamous Cell Carcinomas (SCCs). (A) Bar graph of TCGA data detailing average transcripts per million (TPM) for HORMAD1 gene expression across 23 cancers. A signiﬁcant increase in HORMAD1 expression is observed in squamous cell carcinomas (SCCs) of the cervix (CESC), head and neck (HNSC), esophagus (ESCA), and lung (LUSC) compared to normal adjacent tissue. (B) Immunohistochemical analysis of HOR- MAD1 protein expression in patient biopsy samples of cSCC. HORMAD1 positive control staining (normal human testis) is presented in the upper left panel and normal skin staining is presented in the lower left panel. The remaining panels are cSCC tissues with corresponding magniﬁcation presented in the upper and lower right panels. (C) Quantification of HORMAD1 expression in 18 cSCC patient biopsy samples compared to normal skin and human testis. (D) Bar graph detailing HORMAD1 protein expression in each patient biopsy sample. (E) Qualitative expression of HORMAD1 in cell

Hormad1 Rabbit Polyclonal Antibody, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 93 stars, based on 3 article reviews

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1) Product Images from "Ectopically Expressed Meiosis-Specific Cancer Testis Antigen HORMAD1 Promotes Genomic Instability in Squamous Cell Carcinomas."

Article Title: Ectopically Expressed Meiosis-Specific Cancer Testis Antigen HORMAD1 Promotes Genomic Instability in Squamous Cell Carcinomas.

Journal: Cells

doi: 10.3390/cells12121627

Figure 1. Signiﬁcant increase in the ectopic expression of HORMAD1 in Squamous Cell Carcinomas (SCCs). (A) Bar graph of TCGA data detailing average transcripts per million (TPM) for HORMAD1 gene expression across 23 cancers. A signiﬁcant increase in HORMAD1 expression is observed in squamous cell carcinomas (SCCs) of the cervix (CESC), head and neck (HNSC), esophagus (ESCA), and lung (LUSC) compared to normal adjacent tissue. (B) Immunohistochemical analysis of HOR- MAD1 protein expression in patient biopsy samples of cSCC. HORMAD1 positive control staining (normal human testis) is presented in the upper left panel and normal skin staining is presented in the lower left panel. The remaining panels are cSCC tissues with corresponding magniﬁcation presented in the upper and lower right panels. (C) Quantification of HORMAD1 expression in 18 cSCC patient biopsy samples compared to normal skin and human testis. (D) Bar graph detailing HORMAD1 protein expression in each patient biopsy sample. (E) Qualitative expression of HORMAD1 in cell

Figure Legend Snippet: Figure 1. Signiﬁcant increase in the ectopic expression of HORMAD1 in Squamous Cell Carcinomas (SCCs). (A) Bar graph of TCGA data detailing average transcripts per million (TPM) for HORMAD1 gene expression across 23 cancers. A signiﬁcant increase in HORMAD1 expression is observed in squamous cell carcinomas (SCCs) of the cervix (CESC), head and neck (HNSC), esophagus (ESCA), and lung (LUSC) compared to normal adjacent tissue. (B) Immunohistochemical analysis of HOR- MAD1 protein expression in patient biopsy samples of cSCC. HORMAD1 positive control staining (normal human testis) is presented in the upper left panel and normal skin staining is presented in the lower left panel. The remaining panels are cSCC tissues with corresponding magniﬁcation presented in the upper and lower right panels. (C) Quantification of HORMAD1 expression in 18 cSCC patient biopsy samples compared to normal skin and human testis. (D) Bar graph detailing HORMAD1 protein expression in each patient biopsy sample. (E) Qualitative expression of HORMAD1 in cell

Techniques Used: Expressing, Gene Expression, Immunohistochemical staining, Positive Control, Staining

Figure 2. HORMAD1 expression inﬂuences DNA damage and genomic instability in the cSCC cell line, A431. (A) shRNA-mediated knockdown of HORMAD1 (shHORMAD1) results in increased γH2AX staining (red) indicating high levels DSBs in cells counterstained with DAPI (blue), while overexpression of HORMAD1 (HORMAD1 OE) exhibits minimal γH2AX staining compared to non-silencing CTL cells. Corresponding representative immunoﬂuorescent γH2AX staining for CTL, shHORMAD1 and HORMAD OE cells. Scale bars represent 50 µm. (B) γH2AX (magenta) staining separated into 3 staining types corresponding to the degree of DNA damage: type 1, low DNA damage; type 2, high DNA damage; and type 3, preapoptotic cells (upper panel). Magniﬁcation 1000×. When percent positive γH2AX cells are separated into respective types, shHORMAD1-treated cells display high degree of type 2–3 γH2AX staining (high DNA damage and preapoptotic cells), whereas HORMAD1 OE cells have low levels of DNA damage demonstrated primarily by type 1 γH2AX staining. (C) shHORMAD1 cells exhibit increased genomic instability as indicated by an elevated number of chromatin bridges (arrows, magniﬁcation 1000×) in anaphase and cytokinesis, and a signiﬁcant increase in (D) micronuclei formation (arrows) in cells, nucleic acid stained with cytochalasin B (2 µg/mL) (green). A decrease in chromatin bridge and micronuclei formation was found in HORMAD1 OE cells. Scale bars represent 100 µm. Values are means ± SEM, n = 3, **** p > 0.0001, *** p > 0.001, ** p > 0.01, * p > 0.1, ns (not signiﬁcant); SCC (squamous cell carcinoma); OE (overexpression).

Figure Legend Snippet: Figure 2. HORMAD1 expression inﬂuences DNA damage and genomic instability in the cSCC cell line, A431. (A) shRNA-mediated knockdown of HORMAD1 (shHORMAD1) results in increased γH2AX staining (red) indicating high levels DSBs in cells counterstained with DAPI (blue), while overexpression of HORMAD1 (HORMAD1 OE) exhibits minimal γH2AX staining compared to non-silencing CTL cells. Corresponding representative immunoﬂuorescent γH2AX staining for CTL, shHORMAD1 and HORMAD OE cells. Scale bars represent 50 µm. (B) γH2AX (magenta) staining separated into 3 staining types corresponding to the degree of DNA damage: type 1, low DNA damage; type 2, high DNA damage; and type 3, preapoptotic cells (upper panel). Magniﬁcation 1000×. When percent positive γH2AX cells are separated into respective types, shHORMAD1-treated cells display high degree of type 2–3 γH2AX staining (high DNA damage and preapoptotic cells), whereas HORMAD1 OE cells have low levels of DNA damage demonstrated primarily by type 1 γH2AX staining. (C) shHORMAD1 cells exhibit increased genomic instability as indicated by an elevated number of chromatin bridges (arrows, magniﬁcation 1000×) in anaphase and cytokinesis, and a signiﬁcant increase in (D) micronuclei formation (arrows) in cells, nucleic acid stained with cytochalasin B (2 µg/mL) (green). A decrease in chromatin bridge and micronuclei formation was found in HORMAD1 OE cells. Scale bars represent 100 µm. Values are means ± SEM, n = 3, **** p > 0.0001, *** p > 0.001, ** p > 0.01, * p > 0.1, ns (not signiﬁcant); SCC (squamous cell carcinoma); OE (overexpression).

Techniques Used: Expressing, shRNA, Knockdown, Staining, Over Expression

Figure 3. Depletion of HORMAD1 leads to decreased proliferation and survival. (A) Percent of Ki67 positive cells (red) in control non-silencing cells (CTL), shHORMAD1 and HORMAD1 OE A431 cells 24 h following plating. Nuclei counterstained with DAPI (blue). Scale bars represent 50 µm. (B) Consistent with Ki67 staining, proliferation of shHORMAD1 cells signiﬁcantly decreased 24, 48, and 72 h after plating. (C) Survival/clonogenic assay results complement proliferation results, shHORMAD1 cells formed few colonies, while HORMAD1 OE cells formed signiﬁcantly more colonies than CTL cells. Values are means ± SEM, n = 3, ** p > 0.01, * p > 0.1, ns (not signiﬁcant), SCC (squamous cell carcinoma); OE (overexpression).

Figure Legend Snippet: Figure 3. Depletion of HORMAD1 leads to decreased proliferation and survival. (A) Percent of Ki67 positive cells (red) in control non-silencing cells (CTL), shHORMAD1 and HORMAD1 OE A431 cells 24 h following plating. Nuclei counterstained with DAPI (blue). Scale bars represent 50 µm. (B) Consistent with Ki67 staining, proliferation of shHORMAD1 cells signiﬁcantly decreased 24, 48, and 72 h after plating. (C) Survival/clonogenic assay results complement proliferation results, shHORMAD1 cells formed few colonies, while HORMAD1 OE cells formed signiﬁcantly more colonies than CTL cells. Values are means ± SEM, n = 3, ** p > 0.01, * p > 0.1, ns (not signiﬁcant), SCC (squamous cell carcinoma); OE (overexpression).

Techniques Used: Control, Staining, Clonogenic Assay, Over Expression

Figure 4. HORMAD1 expression provides protection/resistance against DNA damage following etoposide treatment. (A) Quantitative immunoﬂuorescence cell count analysis documenting percent γH2AX staining in non-silencing CTL cells, shHORMAD1 and HORMAD1 OE A431 cells treated with 1 µM etoposide (24 and 72 h panels). At 24 and 72 h following etoposide treatment, shHORMAD1 cells had a signiﬁcantly higher percentage of γH2AX positive cells, while HORMAD1 OE cells had a signiﬁcantly lower percentage of γH2AX positive cells in comparison to CTL. (B) Distribution of percent γH2AX positive cells by corresponding DNA damage type (type 1—low, type 2—high, type 3—preapoptotic, based on γH2AX staining pattern) in cells treated with 1 µM etoposide for 24 h. (C) Percentage of centrosome ampliﬁcation, a marker of genomic instability, indicated by pericentrin immunoﬂuorescence staining (red) in cells treated with etoposide for 72 h. Nuclei counterstained with DAPI (blue). shHORMAD1 cells exhibit centrosome ampliﬁcation, whereas HORMAD1 OE cells demonstrate a signiﬁcantly lower percentage ampliﬁcation compared to CTL. Example images are presented. Magniﬁcation 1000×. (D) Proliferation assays evaluating cell number over 72 h in untreated and etoposide-treated A431 cells. Untreated shHORMAD1 cells exhibit decreased proliferation that is further inhibited following etoposide treatment. Proliferation of HORMAD1 OE cells is minimally affected by etoposide treatment. (E) Clonogenic assays measuring cell survival in untreated and in 1 µM etoposide treated cells over 7–10 days. Values are means ± SEM, n = 3, **** p > 0.0001, *** p > 0.001, * p > 0.1, ns (not signiﬁcant); SCC (squamous cell carcinoma); OE (overexpression).

Figure Legend Snippet: Figure 4. HORMAD1 expression provides protection/resistance against DNA damage following etoposide treatment. (A) Quantitative immunoﬂuorescence cell count analysis documenting percent γH2AX staining in non-silencing CTL cells, shHORMAD1 and HORMAD1 OE A431 cells treated with 1 µM etoposide (24 and 72 h panels). At 24 and 72 h following etoposide treatment, shHORMAD1 cells had a signiﬁcantly higher percentage of γH2AX positive cells, while HORMAD1 OE cells had a signiﬁcantly lower percentage of γH2AX positive cells in comparison to CTL. (B) Distribution of percent γH2AX positive cells by corresponding DNA damage type (type 1—low, type 2—high, type 3—preapoptotic, based on γH2AX staining pattern) in cells treated with 1 µM etoposide for 24 h. (C) Percentage of centrosome ampliﬁcation, a marker of genomic instability, indicated by pericentrin immunoﬂuorescence staining (red) in cells treated with etoposide for 72 h. Nuclei counterstained with DAPI (blue). shHORMAD1 cells exhibit centrosome ampliﬁcation, whereas HORMAD1 OE cells demonstrate a signiﬁcantly lower percentage ampliﬁcation compared to CTL. Example images are presented. Magniﬁcation 1000×. (D) Proliferation assays evaluating cell number over 72 h in untreated and etoposide-treated A431 cells. Untreated shHORMAD1 cells exhibit decreased proliferation that is further inhibited following etoposide treatment. Proliferation of HORMAD1 OE cells is minimally affected by etoposide treatment. (E) Clonogenic assays measuring cell survival in untreated and in 1 µM etoposide treated cells over 7–10 days. Values are means ± SEM, n = 3, **** p > 0.0001, *** p > 0.001, * p > 0.1, ns (not signiﬁcant); SCC (squamous cell carcinoma); OE (overexpression).

Techniques Used: Expressing, Cell Counting, Staining, Comparison, Marker, Over Expression

Figure 5. STRA8 is ectopically expressed in SCCs and its inhibition downregulates HORMAD1. (A) Bar graph of TCGA analysis documenting average transcripts per million (TPM) for STRA8 gene expression across 23 cancers. Consistent with HORMAD1 expression, STRA8 is signiﬁcantly upregulated in squamous cell carcinomas (SCCs) of the cervix (CESC), head and neck (HNSC), esophagus (ESCA), and lung (LUSC) compared to normal adjacent tissue. (B) Immunohistochemical analysis of STRA8 protein expression in patient biopsy samples of cSCCs. STRA8 positive control (normal human testis) is presented in the upper left panel and normal skin biopsy staining is presented in the lower left panel (scale bars represent 50 µm). The remaining panels show STRA8 staining in cSCC tissues with respective magniﬁcation (red square in middle panels; scale bars represent 250 µm) presented in upper and lower right panels (scale bars 50 µm). (C) Quantiﬁcation of STRA8 expression in 18 cSCC patient biopsy samples compared to normal skin. (D) Bar graph detailing STRA8 protein expression in each patient biopsy sample in our patient cohort (C). (E) Immunoblot representing the diminished expression of HORMAD1 protein following shRNA-mediated knockdown of STRA8 (construct 1—Figure S1D) in A431 cells. (F) Cell proliferation results over 72 h for CTL, shSTRA8, and STRA8 OE A431 cells in the presence or absence of 1 µM etoposide treatment. Values are means ± SEM, n = 3, **** p > 0.0001, *** p > 0.001, ** p > 0.01, * p > 0.1. OE (overexpression);

Figure Legend Snippet: Figure 5. STRA8 is ectopically expressed in SCCs and its inhibition downregulates HORMAD1. (A) Bar graph of TCGA analysis documenting average transcripts per million (TPM) for STRA8 gene expression across 23 cancers. Consistent with HORMAD1 expression, STRA8 is signiﬁcantly upregulated in squamous cell carcinomas (SCCs) of the cervix (CESC), head and neck (HNSC), esophagus (ESCA), and lung (LUSC) compared to normal adjacent tissue. (B) Immunohistochemical analysis of STRA8 protein expression in patient biopsy samples of cSCCs. STRA8 positive control (normal human testis) is presented in the upper left panel and normal skin biopsy staining is presented in the lower left panel (scale bars represent 50 µm). The remaining panels show STRA8 staining in cSCC tissues with respective magniﬁcation (red square in middle panels; scale bars represent 250 µm) presented in upper and lower right panels (scale bars 50 µm). (C) Quantiﬁcation of STRA8 expression in 18 cSCC patient biopsy samples compared to normal skin. (D) Bar graph detailing STRA8 protein expression in each patient biopsy sample in our patient cohort (C). (E) Immunoblot representing the diminished expression of HORMAD1 protein following shRNA-mediated knockdown of STRA8 (construct 1—Figure S1D) in A431 cells. (F) Cell proliferation results over 72 h for CTL, shSTRA8, and STRA8 OE A431 cells in the presence or absence of 1 µM etoposide treatment. Values are means ± SEM, n = 3, **** p > 0.0001, *** p > 0.001, ** p > 0.01, * p > 0.1. OE (overexpression);

Techniques Used: Inhibition, Gene Expression, Expressing, Immunohistochemical staining, Positive Control, Staining, Western Blot, shRNA, Knockdown, Construct, Over Expression

Figure 6. Differential expression analysis of RNA-seq data from etoposide-treated and untreated A431 cells. (A) Principal component analysis (PCA) plot depicts clusters of triplicate samples based on similarities in the cells. (B) Signiﬁcantly downregulated DNA repair genes in shRNA-mediated knockdown of HORMAD1 in A431 cells (shHORMAD1 or shH1) or HORMAD1 overexpression (HORMAD1 OE or H1OE). (C) GO BP analysis of upregulated differentially expressed genes (DEGs) in A431 shH1 cells compared to control A431 cells. (D) Signiﬁcantly upregulated DNA repair genes in lentiviral-mediated HORMAD1 overexpressed cells (H1OE), ns (not signiﬁcant). Values are means ± SEM, n = 3, *** p > 0.001, ** p > 0.01.

Figure Legend Snippet: Figure 6. Differential expression analysis of RNA-seq data from etoposide-treated and untreated A431 cells. (A) Principal component analysis (PCA) plot depicts clusters of triplicate samples based on similarities in the cells. (B) Signiﬁcantly downregulated DNA repair genes in shRNA-mediated knockdown of HORMAD1 in A431 cells (shHORMAD1 or shH1) or HORMAD1 overexpression (HORMAD1 OE or H1OE). (C) GO BP analysis of upregulated differentially expressed genes (DEGs) in A431 shH1 cells compared to control A431 cells. (D) Signiﬁcantly upregulated DNA repair genes in lentiviral-mediated HORMAD1 overexpressed cells (H1OE), ns (not signiﬁcant). Values are means ± SEM, n = 3, *** p > 0.001, ** p > 0.01.

Techniques Used: Quantitative Proteomics, RNA Sequencing, shRNA, Knockdown, Over Expression, Control

Image Search Results

Journal: Cells

Article Title: Ectopically Expressed Meiosis-Specific Cancer Testis Antigen HORMAD1 Promotes Genomic Instability in Squamous Cell Carcinomas.

doi: 10.3390/cells12121627

Figure Lengend Snippet: Figure 1. Signiﬁcant increase in the ectopic expression of HORMAD1 in Squamous Cell Carcinomas (SCCs). (A) Bar graph of TCGA data detailing average transcripts per million (TPM) for HORMAD1 gene expression across 23 cancers. A signiﬁcant increase in HORMAD1 expression is observed in squamous cell carcinomas (SCCs) of the cervix (CESC), head and neck (HNSC), esophagus (ESCA), and lung (LUSC) compared to normal adjacent tissue. (B) Immunohistochemical analysis of HOR- MAD1 protein expression in patient biopsy samples of cSCC. HORMAD1 positive control staining (normal human testis) is presented in the upper left panel and normal skin staining is presented in the lower left panel. The remaining panels are cSCC tissues with corresponding magniﬁcation presented in the upper and lower right panels. (C) Quantification of HORMAD1 expression in 18 cSCC patient biopsy samples compared to normal skin and human testis. (D) Bar graph detailing HORMAD1 protein expression in each patient biopsy sample. (E) Qualitative expression of HORMAD1 in cell

Article Snippet: Samples were incubated with HORMAD1 rabbit polyclonal antibody (Novus Biologicals, Centennial, CO, USA, NBP1-85401) or STRA8 (Abcam, ab49602; RRID:AB_945678) at a dilution of 1:500.

Techniques: Expressing, Gene Expression, Immunohistochemical staining, Positive Control, Staining

Journal: Cells

Article Title: Ectopically Expressed Meiosis-Specific Cancer Testis Antigen HORMAD1 Promotes Genomic Instability in Squamous Cell Carcinomas.

doi: 10.3390/cells12121627

Figure Lengend Snippet: Figure 2. HORMAD1 expression inﬂuences DNA damage and genomic instability in the cSCC cell line, A431. (A) shRNA-mediated knockdown of HORMAD1 (shHORMAD1) results in increased γH2AX staining (red) indicating high levels DSBs in cells counterstained with DAPI (blue), while overexpression of HORMAD1 (HORMAD1 OE) exhibits minimal γH2AX staining compared to non-silencing CTL cells. Corresponding representative immunoﬂuorescent γH2AX staining for CTL, shHORMAD1 and HORMAD OE cells. Scale bars represent 50 µm. (B) γH2AX (magenta) staining separated into 3 staining types corresponding to the degree of DNA damage: type 1, low DNA damage; type 2, high DNA damage; and type 3, preapoptotic cells (upper panel). Magniﬁcation 1000×. When percent positive γH2AX cells are separated into respective types, shHORMAD1-treated cells display high degree of type 2–3 γH2AX staining (high DNA damage and preapoptotic cells), whereas HORMAD1 OE cells have low levels of DNA damage demonstrated primarily by type 1 γH2AX staining. (C) shHORMAD1 cells exhibit increased genomic instability as indicated by an elevated number of chromatin bridges (arrows, magniﬁcation 1000×) in anaphase and cytokinesis, and a signiﬁcant increase in (D) micronuclei formation (arrows) in cells, nucleic acid stained with cytochalasin B (2 µg/mL) (green). A decrease in chromatin bridge and micronuclei formation was found in HORMAD1 OE cells. Scale bars represent 100 µm. Values are means ± SEM, n = 3, **** p > 0.0001, *** p > 0.001, ** p > 0.01, * p > 0.1, ns (not signiﬁcant); SCC (squamous cell carcinoma); OE (overexpression).

Techniques: Expressing, shRNA, Knockdown, Staining, Over Expression

Journal: Cells

Article Title: Ectopically Expressed Meiosis-Specific Cancer Testis Antigen HORMAD1 Promotes Genomic Instability in Squamous Cell Carcinomas.

doi: 10.3390/cells12121627

Figure Lengend Snippet: Figure 3. Depletion of HORMAD1 leads to decreased proliferation and survival. (A) Percent of Ki67 positive cells (red) in control non-silencing cells (CTL), shHORMAD1 and HORMAD1 OE A431 cells 24 h following plating. Nuclei counterstained with DAPI (blue). Scale bars represent 50 µm. (B) Consistent with Ki67 staining, proliferation of shHORMAD1 cells signiﬁcantly decreased 24, 48, and 72 h after plating. (C) Survival/clonogenic assay results complement proliferation results, shHORMAD1 cells formed few colonies, while HORMAD1 OE cells formed signiﬁcantly more colonies than CTL cells. Values are means ± SEM, n = 3, ** p > 0.01, * p > 0.1, ns (not signiﬁcant), SCC (squamous cell carcinoma); OE (overexpression).

Techniques: Control, Staining, Clonogenic Assay, Over Expression

Journal: Cells

Article Title: Ectopically Expressed Meiosis-Specific Cancer Testis Antigen HORMAD1 Promotes Genomic Instability in Squamous Cell Carcinomas.

doi: 10.3390/cells12121627

Figure Lengend Snippet: Figure 4. HORMAD1 expression provides protection/resistance against DNA damage following etoposide treatment. (A) Quantitative immunoﬂuorescence cell count analysis documenting percent γH2AX staining in non-silencing CTL cells, shHORMAD1 and HORMAD1 OE A431 cells treated with 1 µM etoposide (24 and 72 h panels). At 24 and 72 h following etoposide treatment, shHORMAD1 cells had a signiﬁcantly higher percentage of γH2AX positive cells, while HORMAD1 OE cells had a signiﬁcantly lower percentage of γH2AX positive cells in comparison to CTL. (B) Distribution of percent γH2AX positive cells by corresponding DNA damage type (type 1—low, type 2—high, type 3—preapoptotic, based on γH2AX staining pattern) in cells treated with 1 µM etoposide for 24 h. (C) Percentage of centrosome ampliﬁcation, a marker of genomic instability, indicated by pericentrin immunoﬂuorescence staining (red) in cells treated with etoposide for 72 h. Nuclei counterstained with DAPI (blue). shHORMAD1 cells exhibit centrosome ampliﬁcation, whereas HORMAD1 OE cells demonstrate a signiﬁcantly lower percentage ampliﬁcation compared to CTL. Example images are presented. Magniﬁcation 1000×. (D) Proliferation assays evaluating cell number over 72 h in untreated and etoposide-treated A431 cells. Untreated shHORMAD1 cells exhibit decreased proliferation that is further inhibited following etoposide treatment. Proliferation of HORMAD1 OE cells is minimally affected by etoposide treatment. (E) Clonogenic assays measuring cell survival in untreated and in 1 µM etoposide treated cells over 7–10 days. Values are means ± SEM, n = 3, **** p > 0.0001, *** p > 0.001, * p > 0.1, ns (not signiﬁcant); SCC (squamous cell carcinoma); OE (overexpression).

Techniques: Expressing, Cell Counting, Staining, Comparison, Marker, Over Expression

Journal: Cells

Article Title: Ectopically Expressed Meiosis-Specific Cancer Testis Antigen HORMAD1 Promotes Genomic Instability in Squamous Cell Carcinomas.

doi: 10.3390/cells12121627

Figure Lengend Snippet: Figure 5. STRA8 is ectopically expressed in SCCs and its inhibition downregulates HORMAD1. (A) Bar graph of TCGA analysis documenting average transcripts per million (TPM) for STRA8 gene expression across 23 cancers. Consistent with HORMAD1 expression, STRA8 is signiﬁcantly upregulated in squamous cell carcinomas (SCCs) of the cervix (CESC), head and neck (HNSC), esophagus (ESCA), and lung (LUSC) compared to normal adjacent tissue. (B) Immunohistochemical analysis of STRA8 protein expression in patient biopsy samples of cSCCs. STRA8 positive control (normal human testis) is presented in the upper left panel and normal skin biopsy staining is presented in the lower left panel (scale bars represent 50 µm). The remaining panels show STRA8 staining in cSCC tissues with respective magniﬁcation (red square in middle panels; scale bars represent 250 µm) presented in upper and lower right panels (scale bars 50 µm). (C) Quantiﬁcation of STRA8 expression in 18 cSCC patient biopsy samples compared to normal skin. (D) Bar graph detailing STRA8 protein expression in each patient biopsy sample in our patient cohort (C). (E) Immunoblot representing the diminished expression of HORMAD1 protein following shRNA-mediated knockdown of STRA8 (construct 1—Figure S1D) in A431 cells. (F) Cell proliferation results over 72 h for CTL, shSTRA8, and STRA8 OE A431 cells in the presence or absence of 1 µM etoposide treatment. Values are means ± SEM, n = 3, **** p > 0.0001, *** p > 0.001, ** p > 0.01, * p > 0.1. OE (overexpression);

Techniques: Inhibition, Gene Expression, Expressing, Immunohistochemical staining, Positive Control, Staining, Western Blot, shRNA, Knockdown, Construct, Over Expression

Journal: Cells

Article Title: Ectopically Expressed Meiosis-Specific Cancer Testis Antigen HORMAD1 Promotes Genomic Instability in Squamous Cell Carcinomas.

doi: 10.3390/cells12121627

Figure Lengend Snippet: Figure 6. Differential expression analysis of RNA-seq data from etoposide-treated and untreated A431 cells. (A) Principal component analysis (PCA) plot depicts clusters of triplicate samples based on similarities in the cells. (B) Signiﬁcantly downregulated DNA repair genes in shRNA-mediated knockdown of HORMAD1 in A431 cells (shHORMAD1 or shH1) or HORMAD1 overexpression (HORMAD1 OE or H1OE). (C) GO BP analysis of upregulated differentially expressed genes (DEGs) in A431 shH1 cells compared to control A431 cells. (D) Signiﬁcantly upregulated DNA repair genes in lentiviral-mediated HORMAD1 overexpressed cells (H1OE), ns (not signiﬁcant). Values are means ± SEM, n = 3, *** p > 0.001, ** p > 0.01.

Techniques: Quantitative Proteomics, RNA Sequencing, shRNA, Knockdown, Over Expression, Control

HORMAD1 localization in a zebra finch pachytene spermatocyte ( A – D ). Locations of proteins SYCP3 (green) and HORMAD1 (white) are shown. Chromatin is stained with DAPI (blue). GRC: germline-restricted chromosome. The white circles point to GRCs. Scale bar = 5 µm.

Journal: Animals : an Open Access Journal from MDPI

Article Title: The Germline-Restricted Chromosome of Male Zebra Finches in Meiotic Prophase I: A Proteinaceous Scaffold and Chromatin Modifications

doi: 10.3390/ani14223246

Figure Lengend Snippet: HORMAD1 localization in a zebra finch pachytene spermatocyte ( A – D ). Locations of proteins SYCP3 (green) and HORMAD1 (white) are shown. Chromatin is stained with DAPI (blue). GRC: germline-restricted chromosome. The white circles point to GRCs. Scale bar = 5 µm.

Article Snippet: Immunostaining was performed using an anti-SYCP3 (synaptonemal complex protein 3) rabbit polyclonal antibody (Abcam, Cambridge, UK, ab15093), rabbit polyclonal anti-HORMAD1 antibody (Proteintech, Rosemont, IL, USA, 13917-1-AP), rabbit polyclonal anti-RPA32/RPA2 antibody (RPA; Abcam, ab10359), mouse polyclonal anti-RAD51 antibody (Abcam, ab88572), rabbit polyclonal anti-histone H3 antibody (tri methyl K27) (H3K27me3; Abcam, ab195477), mouse monoclonal anti-RNA polymerase II antibody (RNAP II; Abcam, ab5408), mouse monoclonal anti-gammaH2A.X antibody (phospho S139) [9F3] (γH2AFX; Abcam, ab26350), mouse monoclonal anti-SUMO-1 antibody (Zymed, San Francisco, CA, USA #33-2400), mouse monoclonal anti-ubiquityl histone H2A antibody (ubiH2A; Millipore, Billerica, MA, USA, #05-678), rabbit polyclonal anti-H3K9me3 antibody (Abcam, ab8898), rat monoclonal anti-histone H3 antibody (phospho S28) (H3S28ph; Abcam, ab10543), and a human anti-centromere antibody (CREST [calcinosis, Raynaud phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia]; #90C-CS1058, Fitzgerald Industries International, Concord, MA, USA).

Techniques: Staining

Dynamics of GRC structure at prophase I of zebra finch meiosis. Schematic representations of the structure and behavior of meiotic chromosomes are depicted. As the scheme is simplified, it does not capture the chromatin’s loop organization. Abbreviations: GRC, germline-restricted chromosome; SC, synaptonemal complex; AE, axial element; LE, lateral element of SC; TF, transverse filament of central space of SC; as, asynapsis area; ds, desynapsis area. In AEs/LEs of SCs: HORMAD1 (blue dots), cohesins (red dots), and SYCP3 (green dots). Typically, the GRC was found to create a distinct chromatin domain at the nuclear periphery during prophase I ( A – D ). ( A ) Leptotene. At this stage, core proteins (cohesins, HORMAD1 and SYCP3) ( A′ ) are loaded into the chromosome axis; therefore, the nascent proteinaceous scaffolds of the AEs of autosomes and GRC can be observed. ( B ) Zygotene. Chromosomes form a bouquet. Autosomal AEs arise. Two AEs of homologous chromosomes align with each other and begin to form SC segments. SC regions either lack HORMAD1 or contain rare stand-alone signals. Unlike autosomes, where protein assembly of AEs was completed, loading of core proteins continues into the AE of the GRC (protein loading delay) ( B′ ). Asynaptic regions and some regions of the GRC univalent contain a large amount of HORMAD1. ( C ) Pachytene. Autosomes are fully synapsed. Loading of core proteins into the GRC AE is almost complete. Sometimes, some regions of the GRC remain without loaded core proteins (fragmented GRC AEs or GRC with gaps) ( C′ ). Unlike autosomes, the GRC univalent has a lot of HORMAD1. ( D ) Diplotene. Chromosomes are desynapsed. The GRC and desynapsing axes of autosomes undergo protein disassembly. Desynapsing segments of autosomes and GRC are enriched with HORMAD1. ( E ) Explanatory diagram.

Journal: Animals : an Open Access Journal from MDPI

Article Title: The Germline-Restricted Chromosome of Male Zebra Finches in Meiotic Prophase I: A Proteinaceous Scaffold and Chromatin Modifications

doi: 10.3390/ani14223246

Figure Lengend Snippet: Dynamics of GRC structure at prophase I of zebra finch meiosis. Schematic representations of the structure and behavior of meiotic chromosomes are depicted. As the scheme is simplified, it does not capture the chromatin’s loop organization. Abbreviations: GRC, germline-restricted chromosome; SC, synaptonemal complex; AE, axial element; LE, lateral element of SC; TF, transverse filament of central space of SC; as, asynapsis area; ds, desynapsis area. In AEs/LEs of SCs: HORMAD1 (blue dots), cohesins (red dots), and SYCP3 (green dots). Typically, the GRC was found to create a distinct chromatin domain at the nuclear periphery during prophase I ( A – D ). ( A ) Leptotene. At this stage, core proteins (cohesins, HORMAD1 and SYCP3) ( A′ ) are loaded into the chromosome axis; therefore, the nascent proteinaceous scaffolds of the AEs of autosomes and GRC can be observed. ( B ) Zygotene. Chromosomes form a bouquet. Autosomal AEs arise. Two AEs of homologous chromosomes align with each other and begin to form SC segments. SC regions either lack HORMAD1 or contain rare stand-alone signals. Unlike autosomes, where protein assembly of AEs was completed, loading of core proteins continues into the AE of the GRC (protein loading delay) ( B′ ). Asynaptic regions and some regions of the GRC univalent contain a large amount of HORMAD1. ( C ) Pachytene. Autosomes are fully synapsed. Loading of core proteins into the GRC AE is almost complete. Sometimes, some regions of the GRC remain without loaded core proteins (fragmented GRC AEs or GRC with gaps) ( C′ ). Unlike autosomes, the GRC univalent has a lot of HORMAD1. ( D ) Diplotene. Chromosomes are desynapsed. The GRC and desynapsing axes of autosomes undergo protein disassembly. Desynapsing segments of autosomes and GRC are enriched with HORMAD1. ( E ) Explanatory diagram.

Techniques:

Figure 5. HORMAD1 and HORMAD2 accumulate on the lateral elements of synapsed autosomes in Trip13-

Journal: eLife

Article Title: TRIP13 localizes to synapsed chromosomes and functions as a dosage-sensitive regulator of meiosis

doi: 10.7554/elife.92195

Figure Lengend Snippet: Figure 5. HORMAD1 and HORMAD2 accumulate on the lateral elements of synapsed autosomes in Trip13-

Article Snippet: or resource Designation Source or reference Identifiers Additional information Gene (Mus musculus) Trip13 GenBank Gene ID: 69716 Genetic reagent (M. musculus) Trip13tm1.1(KOMP)Vlcg/JMmucd MMRCC MMRRC_050223- UCD Genetic reagent (M. musculus) Hormad1 knockout PMID:21079677; Shin et al., 2010 Rajkovic lab Genetic reagent (M. musculus) Sycp2 knockout PMID:16717126; Yang et al., 2006 Wang lab Genetic reagent (M. musculus) Rec8 knockout PMID:32232159; Guan et al., 2020 Wang lab Genetic reagent (M. musculus) 3×FLAG-Trip13 This paper Wang Lab Genetic reagent (M. musculus) Trip13-3×FLAG This paper Wang Lab Antibody Anti- ACTB (mouse monoclonal) Sigma Cat# A5441, RRID:AB_476744 WB (1:2000) Antibody Anti- centromere (CREST) (human polyclonal) Antibodies Incorporated Cat# 15- 234, RRID:AB_2687472 IF (1:500) Antibody Anti- SYCP1 (rabbit polyclonal) Abcam Cat# ab15090, RRID:AB_301636 IF (1:300) Antibody Anti- SYCP2 (guinea pig polyclonal) PMID:16717126 Custom made IF (1:150) Antibody Anti- SYCP3 (mouse monoclonal) Abcam Cat# ab97672, RRID:AB_10678841 IF (1:500) Antibody Anti- SYCP3 (rabbit polyclonal) ProteinTech Group Cat# 23024- 1- AP, RRID:AB_11232426 IF (1:500), WB (1:2000) Antibody Anti- HORMAD1 (rabbit polyclonal) ProteinTech Group Cat# 13917- 1- AP, RRID:AB_2120844 IF (1:300) Antibody Anti- HORMAD2 (rabbit polyclonal) PMID:19851446; Wojtasz et al., 2009 A gift from Toth lab IF (1:500) Antibody Anti- REC114 (rabbit polyclonal) PMID:31003867; Boekhout et al., 2019 A gift from Keeney Lab IF (1:100) Antibody Anti- REC8 (rabbit polyclonal) Custom made A gift from Mengcheng Luo lab IF (1:200) Antibody Anti- FLAG (mouse monoclonal) Sigma Cat# F3165, RRID:AB_259529 IF (1:300), WB (1:4000) Antibody Anti- SKP1 (rabbit polyclonal) Cell Signaling Cat# 12248S, RRID:AB_2754993 IF (1:200), WB (1:1000) Antibody Anti- TRIP13 (rabbit polyclonal) ProteinTech Group Cat# 19602- 1- AP IF (1:150), WB (1:1000) Chotiner et al. eLife 2023;12:RP92195.

Techniques:

Figure 6. Localization of TRIP13 to the synaptonemal complex (SC) is independent of individual axial element components. (A) Immunofluorescent analysis of TRIP13 in Hormad1-/- spermatocytes from 2-month-old mice. (B) Immunofluorescent analysis of TRIP13 in Rec8-/- spermatocytes from 2-month- old mice. (C) Immunofluorescent analysis of TRIP13 in Sycp2-/- spermatocytes from 2-month-old mice. (D) Immunofluorescent analysis of TRIP13 in Skp1cKO spermatocytes from 2-month-old mice. Scale bars, 10 µm.

Journal: eLife

Article Title: TRIP13 localizes to synapsed chromosomes and functions as a dosage-sensitive regulator of meiosis

doi: 10.7554/elife.92195

Figure Lengend Snippet: Figure 6. Localization of TRIP13 to the synaptonemal complex (SC) is independent of individual axial element components. (A) Immunofluorescent analysis of TRIP13 in Hormad1-/- spermatocytes from 2-month-old mice. (B) Immunofluorescent analysis of TRIP13 in Rec8-/- spermatocytes from 2-month- old mice. (C) Immunofluorescent analysis of TRIP13 in Sycp2-/- spermatocytes from 2-month-old mice. (D) Immunofluorescent analysis of TRIP13 in Skp1cKO spermatocytes from 2-month-old mice. Scale bars, 10 µm.

Techniques:

Figure 7. FLAG-tagged TRIP13 proteins localize correctly and are functional. (A) Western blot analysis of tagged and untagged TRIP13 proteins in testes from P20 wild type (no tag), heterozygous-tagged, and homozygous-tagged males. (B) Immunofluorescence of FLAG-tagged TRIP13 in pachytene spermatocytes from P20 homozygous testes. N-terminal tag, 3×FLAG-Trip13; C-terminal tag, Trip13−3×FLAG. Scale bar, 10 μm. (C) A schematic illustration of TRIP13, SKP1, HORMAD1/2, and the synaptonemal complex. Relative locations of TRIP13 and SKP1 within the synaptonemal complex (SC) are depicted. HORAMD1/2 are retained in synapsed regions in Trip13-deficient or Skp1-deficient spermatocytes.

Journal: eLife

Article Title: TRIP13 localizes to synapsed chromosomes and functions as a dosage-sensitive regulator of meiosis

doi: 10.7554/elife.92195

Figure Lengend Snippet: Figure 7. FLAG-tagged TRIP13 proteins localize correctly and are functional. (A) Western blot analysis of tagged and untagged TRIP13 proteins in testes from P20 wild type (no tag), heterozygous-tagged, and homozygous-tagged males. (B) Immunofluorescence of FLAG-tagged TRIP13 in pachytene spermatocytes from P20 homozygous testes. N-terminal tag, 3×FLAG-Trip13; C-terminal tag, Trip13−3×FLAG. Scale bar, 10 μm. (C) A schematic illustration of TRIP13, SKP1, HORMAD1/2, and the synaptonemal complex. Relative locations of TRIP13 and SKP1 within the synaptonemal complex (SC) are depicted. HORAMD1/2 are retained in synapsed regions in Trip13-deficient or Skp1-deficient spermatocytes.

Techniques: Functional Assay, Western Blot, Immunofluorescence

A, B Y2H assays between IHO1 interactors (this study and , , ) and wild-type (1-574) or modified versions of IHO1. A Schematics show conserved domains and positions of phospho-serines (S) or -threonine (T) or their substitution with alanine (A) in IHO1. B Budding yeast cultures co-transformed with indicated pairs of Y2H baits (top) and preys (left side) are shown after 3 (two left images) or 2 (two right images) days of growth on drop-out plates. X marks bait-prey combinations that were omitted from Y2H due to lack of relevance. C, G Immunostaining in nuclear spread spermatocytes of 13 days postpartum (dpp) ( C ) and adult ( G ) mice. Chromosome axis (SYCP3, C overlay, G) , HORMAD1 ( C, G ) and either ectopically expressed GFP-IHO1 ( C ) or endogenous IHO1 ( G ) were detected. Bars, 10 µm. D Quantification of localization of GFP-tagged IHO1 versions in late zygotene. IHO1 versions: wild type (WT), a mutant missing the last 7 amino acids (C7Δ) and versions where single-letter amino acid code indicates point mutations in positions 569 and 570. Block bars are means. Likelihood-ratio test, ns= P > 0.05, ***= P < 0.001, ****= P < 0.0001. Exact P values: WT vs. C7Δ and AA vs. SA P < 2.2e-16, C7Δ vs. AA P = 0,06547, SA vs. SD P = 9.445e-10, SA vs. SE P = 2.225e-06, AA vs. AS P = 3.217e-09, AS vs. DS P = 4.979e-14, AS vs. ES P = 2.966e-11, AA vs. DD P = 2.599e-09, AA vs. EE P = 0.001003. E Immunoprecipitation (IP) immunoblots from testis extracts of 13 dpp mice. Asterisk and triangles mark unspecific protein band in REC114 blot and isoforms of SYCP3, respectively. Distinct proteins were detected on separate blots. F Schematics summarizing conclusions of panel E. See also related Supplementary Fig. , Supplementary Table and . Source data are provided as a Source Data file.

Journal: Nature Communications

Article Title: Seeding the meiotic DNA break machinery and initiating recombination on chromosome axes

doi: 10.1038/s41467-024-47020-1

Figure Lengend Snippet: A, B Y2H assays between IHO1 interactors (this study and , , ) and wild-type (1-574) or modified versions of IHO1. A Schematics show conserved domains and positions of phospho-serines (S) or -threonine (T) or their substitution with alanine (A) in IHO1. B Budding yeast cultures co-transformed with indicated pairs of Y2H baits (top) and preys (left side) are shown after 3 (two left images) or 2 (two right images) days of growth on drop-out plates. X marks bait-prey combinations that were omitted from Y2H due to lack of relevance. C, G Immunostaining in nuclear spread spermatocytes of 13 days postpartum (dpp) ( C ) and adult ( G ) mice. Chromosome axis (SYCP3, C overlay, G) , HORMAD1 ( C, G ) and either ectopically expressed GFP-IHO1 ( C ) or endogenous IHO1 ( G ) were detected. Bars, 10 µm. D Quantification of localization of GFP-tagged IHO1 versions in late zygotene. IHO1 versions: wild type (WT), a mutant missing the last 7 amino acids (C7Δ) and versions where single-letter amino acid code indicates point mutations in positions 569 and 570. Block bars are means. Likelihood-ratio test, ns= P > 0.05, ***= P < 0.001, ****= P < 0.0001. Exact P values: WT vs. C7Δ and AA vs. SA P < 2.2e-16, C7Δ vs. AA P = 0,06547, SA vs. SD P = 9.445e-10, SA vs. SE P = 2.225e-06, AA vs. AS P = 3.217e-09, AS vs. DS P = 4.979e-14, AS vs. ES P = 2.966e-11, AA vs. DD P = 2.599e-09, AA vs. EE P = 0.001003. E Immunoprecipitation (IP) immunoblots from testis extracts of 13 dpp mice. Asterisk and triangles mark unspecific protein band in REC114 blot and isoforms of SYCP3, respectively. Distinct proteins were detected on separate blots. F Schematics summarizing conclusions of panel E. See also related Supplementary Fig. , Supplementary Table and . Source data are provided as a Source Data file.

Article Snippet: After Western blotting the membrane was blocked in 5% nonfat milk in PBS with 0.1% Tween-20 (PBST) and incubated with primary antibodies overnight at 4 ° C. Rabbit polyclonal anti-HORMAD1 antibodies (Abcam) were used at the concentration 1 μg/μl.

Techniques: Modification, Transformation Assay, Immunostaining, Mutagenesis, Blocking Assay, Immunoprecipitation, Western Blot

A Testis to body weight ratios in adult mice (age 50-120 days). Bars mark means. Two-tailed Welch t-test, ****= P < 0.0001, WT vs. Iho1 C7Δ/C7Δ P = 4.88e-05, Iho1 C7Δ/C7Δ vs. Hormad1 −/− P = 1.71e-07. B , E Immunostaining in nuclear spread leptotene spermatocytes of adult mice. Bars, 10 µm. C , D , F , G Numbers of small MEI4-REC114 co-clusters ( B , E ) and MEI4 intensities in MEI4-REC114 co-clusters ( C , F , data points show median cluster intensities per cell) in spermatocytes of adult mice. Zygo-pachytene ( F , G ) is equivalent to a mix of late-zygotene and early pachytene stages which are indistinguishable in SC-defective backgrounds. Pooled data is shown from 5 ( C , D ) or 2 ( F , G ) mice of each genotype. Bars are medians, n=cell numbers. Two-tailed Mann Whitney U-Test, ns= P > 0.05, *= P < 0.05, ***= P < 0.001, ****= P < 0.0001. Exact P values: ( C , F ), P < 2.2e-16 for all comparisons, ( D ), pre-leptotene, wild type vs. Iho1 C7Δ/C7Δ , P = 1.63e-10, wild type vs. Hormad1 −/− , P = 3.39e-15, wild type vs. Iho1 C7Δ/C7Δ Hormad1 −/− , P = 1.97e-11, leptotene, wild type vs. Iho1 C7Δ/C7Δ , P = 0.04839, wild type vs. Hormad1 −/− , P = 3.57e-5, wild type vs. Iho1 C7Δ/C7Δ Hormad1 −/− , P = 0.782, early zygotene, wild type vs. Iho1 C7Δ/C7Δ , P = 1.84e-7, wild type vs. Hormad1 −/− , P = 1.53e-5, wild type vs. Iho1 C7Δ/C7Δ Hormad1 −/− , P = 3.42e-5, ( G ), pre-leptotene, Spo11 −/− Iho1 +/+ vs. Spo11 −/− Iho1 C7Δ/C7Δ , P < 2.2e-16, Spo11 −/− Hormad1 +/+ vs. Spo11 −/− Hormad1 −/− P = 5.03e-11, leptotene, Spo11 −/− Iho1 +/+ vs. Spo11 −/− Iho1 C7Δ/C7Δ , P = 1.37e-10, Spo11 −/− Hormad1 +/+ vs. Spo11 −/− Hormad1 −/− P = 9.16e-15, early zygotene, Spo11 −/− Iho1 +/+ vs. Spo11 −/− Iho1 C7Δ/C7Δ , P < 2.2e-16, Spo11 −/− Hormad1 +/+ vs. Spo11 −/− Hormad1 −/− P < 2.2e-16, zygo-pachytene, Spo11 −/− Iho1 +/+ vs. Spo11 −/− Iho1 C7Δ/C7Δ , P = 8.4e-7, Spo11 −/− Hormad1 +/+ vs. Spo11 −/− Hormad1 −/− P = 0.0003408. Statistical tests compare samples that were stained and processed in parallel within experimental repeats to reduce technical variability. Thus, Spo11 −/− Iho1 +/+and C7Δ/C7Δ are not directly comparable with Spo11 −/− Hormad1 +/+and−/− due to sample preparation from different colonies on different days ( F , G ). See also related Supplementary Fig. , and Supplementary Tables and . Source data are provided as a Source Data file.

Journal: Nature Communications

Article Title: Seeding the meiotic DNA break machinery and initiating recombination on chromosome axes

doi: 10.1038/s41467-024-47020-1

Figure Lengend Snippet: A Testis to body weight ratios in adult mice (age 50-120 days). Bars mark means. Two-tailed Welch t-test, ****= P < 0.0001, WT vs. Iho1 C7Δ/C7Δ P = 4.88e-05, Iho1 C7Δ/C7Δ vs. Hormad1 −/− P = 1.71e-07. B , E Immunostaining in nuclear spread leptotene spermatocytes of adult mice. Bars, 10 µm. C , D , F , G Numbers of small MEI4-REC114 co-clusters ( B , E ) and MEI4 intensities in MEI4-REC114 co-clusters ( C , F , data points show median cluster intensities per cell) in spermatocytes of adult mice. Zygo-pachytene ( F , G ) is equivalent to a mix of late-zygotene and early pachytene stages which are indistinguishable in SC-defective backgrounds. Pooled data is shown from 5 ( C , D ) or 2 ( F , G ) mice of each genotype. Bars are medians, n=cell numbers. Two-tailed Mann Whitney U-Test, ns= P > 0.05, *= P < 0.05, ***= P < 0.001, ****= P < 0.0001. Exact P values: ( C , F ), P < 2.2e-16 for all comparisons, ( D ), pre-leptotene, wild type vs. Iho1 C7Δ/C7Δ , P = 1.63e-10, wild type vs. Hormad1 −/− , P = 3.39e-15, wild type vs. Iho1 C7Δ/C7Δ Hormad1 −/− , P = 1.97e-11, leptotene, wild type vs. Iho1 C7Δ/C7Δ , P = 0.04839, wild type vs. Hormad1 −/− , P = 3.57e-5, wild type vs. Iho1 C7Δ/C7Δ Hormad1 −/− , P = 0.782, early zygotene, wild type vs. Iho1 C7Δ/C7Δ , P = 1.84e-7, wild type vs. Hormad1 −/− , P = 1.53e-5, wild type vs. Iho1 C7Δ/C7Δ Hormad1 −/− , P = 3.42e-5, ( G ), pre-leptotene, Spo11 −/− Iho1 +/+ vs. Spo11 −/− Iho1 C7Δ/C7Δ , P < 2.2e-16, Spo11 −/− Hormad1 +/+ vs. Spo11 −/− Hormad1 −/− P = 5.03e-11, leptotene, Spo11 −/− Iho1 +/+ vs. Spo11 −/− Iho1 C7Δ/C7Δ , P = 1.37e-10, Spo11 −/− Hormad1 +/+ vs. Spo11 −/− Hormad1 −/− P = 9.16e-15, early zygotene, Spo11 −/− Iho1 +/+ vs. Spo11 −/− Iho1 C7Δ/C7Δ , P < 2.2e-16, Spo11 −/− Hormad1 +/+ vs. Spo11 −/− Hormad1 −/− P < 2.2e-16, zygo-pachytene, Spo11 −/− Iho1 +/+ vs. Spo11 −/− Iho1 C7Δ/C7Δ , P = 8.4e-7, Spo11 −/− Hormad1 +/+ vs. Spo11 −/− Hormad1 −/− P = 0.0003408. Statistical tests compare samples that were stained and processed in parallel within experimental repeats to reduce technical variability. Thus, Spo11 −/− Iho1 +/+and C7Δ/C7Δ are not directly comparable with Spo11 −/− Hormad1 +/+and−/− due to sample preparation from different colonies on different days ( F , G ). See also related Supplementary Fig. , and Supplementary Tables and . Source data are provided as a Source Data file.

Techniques: Two Tailed Test, Immunostaining, MANN-WHITNEY, Staining, Sample Prep

A Radiograph of immunoprecipitated and radioactively labeled SPO11-oligo complexes from testes of 13 dpp juvenile mice. Bar, SPO11-specific signals, asterisk, nonspecific labelling, and arrowhead, immunoglobulin heavy-chain. Radioactive signals were background-corrected ( Iho1 −/− , signal=0) and normalized to corresponding wild type control (1). B Quantification of SPO11-oligo complexes from 13dpp mice. Bars are mean, n=number of mice. Two-tailed paired t-test, ns=P = 0.3419. C – E Immunostaining in nuclear spread early zygotene spermatocytes of adult mice. Bars, 10 µm. F , G Quantification of axis associated DMC1 ( F ), RPA2 ( G ) focus numbers in spermatocytes. Pools of two experiments are shown (one mouse represented each genotype in each experiment). Bars are medians, n=cell numbers. Two-tailed Mann Whitney U-test, ns=P > 0.05, *=P < 0.05, **=P < 0.01, ****= P < 0.0001. Exact P values: ( F ), leptotene, wild type vs. Iho1 C7Δ/C7Δ , P = 0.05, Iho1 C7Δ/C7Δ vs. Hormad1 −/− , P = 0.486, Hormad1 −/− vs. Hormad1 −/− Iho1 C7Δ/C7Δ , P = 0.234, early zygotene, wild type vs. Iho1 C7Δ/C7Δ , P = 1.26e-7, Iho1 C7Δ/C7Δ vs. Hormad1 −/− , P = 7.38e-6, Hormad1 −/− vs. Hormad1 −/− Iho1 C7Δ/C7Δ , P = 0.984, late zygotene, wild type vs. Iho1 C7Δ/C7Δ , P = 9.44e-9, Iho1 C7Δ/C7Δ vs. Hormad1 −/− , P = 1.61e-6, Hormad1 −/− vs. Hormad1 −/− Iho1 C7Δ/C7Δ , P = 0.373, pachytene, wild type vs. Iho1 C7Δ/C7Δ , P = 1.5e-6, ( G ), leptotene, wild type vs. Iho1 C7Δ/C7Δ , P = 0.02417, Iho1 C7Δ/C7Δ vs. Hormad1 −/− , P = 0.01189, Hormad1 −/− vs. Hormad1 −/− Iho1 C7Δ/C7Δ , P = 0.2825, early zygotene, wild type vs. Iho1 C7Δ/C7Δ , P = 2.19e-9, Iho1 C7Δ/C7Δ vs. Hormad1 −/ − , P = 3.17e-8, Hormad1 −/− vs. Hormad1 −/− Iho1 C7Δ/C7Δ , P = 0.2455, late zygotene, wild type vs. Iho1 C7Δ/C7Δ , P < 2.2e-16, Iho1 C7Δ/C7Δ vs. Hormad1 −/− , P < 2.2e-16, Hormad1 −/− vs. Hormad1 −/− Iho1 C7Δ/C7Δ , P = 0.4054, pachytene, wild type vs. Iho1 C7Δ/C7Δ , P = 7.28e-15. See also related Supplementary Fig. . Source data are provided as a Source Data file.

Journal: Nature Communications

Article Title: Seeding the meiotic DNA break machinery and initiating recombination on chromosome axes

doi: 10.1038/s41467-024-47020-1

Figure Lengend Snippet: A Radiograph of immunoprecipitated and radioactively labeled SPO11-oligo complexes from testes of 13 dpp juvenile mice. Bar, SPO11-specific signals, asterisk, nonspecific labelling, and arrowhead, immunoglobulin heavy-chain. Radioactive signals were background-corrected ( Iho1 −/− , signal=0) and normalized to corresponding wild type control (1). B Quantification of SPO11-oligo complexes from 13dpp mice. Bars are mean, n=number of mice. Two-tailed paired t-test, ns=P = 0.3419. C – E Immunostaining in nuclear spread early zygotene spermatocytes of adult mice. Bars, 10 µm. F , G Quantification of axis associated DMC1 ( F ), RPA2 ( G ) focus numbers in spermatocytes. Pools of two experiments are shown (one mouse represented each genotype in each experiment). Bars are medians, n=cell numbers. Two-tailed Mann Whitney U-test, ns=P > 0.05, *=P < 0.05, **=P < 0.01, ****= P < 0.0001. Exact P values: ( F ), leptotene, wild type vs. Iho1 C7Δ/C7Δ , P = 0.05, Iho1 C7Δ/C7Δ vs. Hormad1 −/− , P = 0.486, Hormad1 −/− vs. Hormad1 −/− Iho1 C7Δ/C7Δ , P = 0.234, early zygotene, wild type vs. Iho1 C7Δ/C7Δ , P = 1.26e-7, Iho1 C7Δ/C7Δ vs. Hormad1 −/− , P = 7.38e-6, Hormad1 −/− vs. Hormad1 −/− Iho1 C7Δ/C7Δ , P = 0.984, late zygotene, wild type vs. Iho1 C7Δ/C7Δ , P = 9.44e-9, Iho1 C7Δ/C7Δ vs. Hormad1 −/− , P = 1.61e-6, Hormad1 −/− vs. Hormad1 −/− Iho1 C7Δ/C7Δ , P = 0.373, pachytene, wild type vs. Iho1 C7Δ/C7Δ , P = 1.5e-6, ( G ), leptotene, wild type vs. Iho1 C7Δ/C7Δ , P = 0.02417, Iho1 C7Δ/C7Δ vs. Hormad1 −/− , P = 0.01189, Hormad1 −/− vs. Hormad1 −/− Iho1 C7Δ/C7Δ , P = 0.2825, early zygotene, wild type vs. Iho1 C7Δ/C7Δ , P = 2.19e-9, Iho1 C7Δ/C7Δ vs. Hormad1 −/ − , P = 3.17e-8, Hormad1 −/− vs. Hormad1 −/− Iho1 C7Δ/C7Δ , P = 0.2455, late zygotene, wild type vs. Iho1 C7Δ/C7Δ , P < 2.2e-16, Iho1 C7Δ/C7Δ vs. Hormad1 −/− , P < 2.2e-16, Hormad1 −/− vs. Hormad1 −/− Iho1 C7Δ/C7Δ , P = 0.4054, pachytene, wild type vs. Iho1 C7Δ/C7Δ , P = 7.28e-15. See also related Supplementary Fig. . Source data are provided as a Source Data file.

Techniques: Immunoprecipitation, Labeling, Two Tailed Test, Immunostaining, MANN-WHITNEY

A, B, D Numbers of small MEI4-REC114 co-clusters ( A ), MEI4 intensities in MEI4-REC114 co-clusters ( B , data points show median cluster intensities per cell), and DMC1 focus numbers ( D ) in spermatocytes of adult mice. Zygo-pachytene ( D) , only in SC-defective backgrounds) is equivalent to a mix of late-zygotene and early pachytene stages which are indistinguishable if SC is defective. Pooled data is shown from 6 ( A, B ) or 2 ( D ) mice of each genotype. Bars are medians, n=cell numbers. Two-tailed Mann Whitney U-Test, ns= P > 0.05, *= P < 0.05, **=P < 0.01, ***= P < 0.001, ****= P < 0.0001. Exact P values: ( A ), wild type (wt) versus Ankrd31 −/− pre-leptotene P = 5.35e-13, early zygotene P = 0.0001596, all the others P < 2.2e-16, ( B ), all comparisons in pre-leptotene and leptotene P < 2.2e-16, early zygotene, wt vs. Ankrd31 −/− P = 0.3047, Ankrd31 −/− vs. Iho1 C7Δ/C7Δ P = 0.001186, Ankrd31 −/− vs. Hormad1 −/− P = 7.19e-5, ( D ), leptotene, wt vs. Ankrd31 −/− P = 1.07e-6, Ankrd31 −/− vs. Iho1 C7Δ/C7Δ P = 0.01621, Iho1 C7Δ/C7Δ vs. Ankrd31 −/− Iho1 C7Δ/C7Δ P = 5.28e-5, Ankrd31 −/− vs. Hormad1 −/− P = 0.1945, Hormad1 −/− vs. Ankrd31 −/− Hormad1 −/− P = 0.175, early zygotene, wt vs. Ankrd31 −/− P = 5.82e-10, Ankrd31 −/− vs. Iho1 C7Δ/C7Δ P = 0.00041, Iho1 C7Δ/C7Δ vs. Ankrd31 −/− Iho1 C7Δ/C7Δ P = 1.47e-13, Ankrd31 −/− vs. Hormad1 −/− P = 0.0002882, Hormad1 −/− vs. Ankrd31 −/− Hormad1 −/− P < 2.2e-16, late zygotene, wt vs. Ankrd31 −/− P = 0.0004372, Ankrd31 −/− vs. Iho1 C7Δ/C7Δ P = 1.48e-13, Iho1 C7Δ/C7Δ vs. Ankrd31 −/− Iho1 C7Δ/C7Δ P = 5.35e-16, Ankrd31 −/− vs. Hormad1 −/− P < 2.2e-16, Hormad1 −/− vs. Ankrd31 −/− Hormad1 −/− P = 2.49e-11, early pachytene, wt vs. Ankrd31 −/− P = 0.0001234, Ankrd31 −/− vs. Iho1 C7Δ/C7Δ P = 1.1e-10. C Immunostaining in leptotene spermatocytes from adult mice. Bars, 10 µm. E Radiograph of immunoprecipitated and radioactively labeled SPO11-oligo complexes from testes of adult mice. Bar, SPO11-specific signal, asterisk, nonspecific labelling, and arrowhead, immunoglobulin heavy-chain. Radioactive signals were background-corrected ( Iho1 −/− , signal=0) and normalized to wild-type control (1). Means and standard deviations are from n = 2 biological replicates. F Schematic summary of phenotypes caused by the disruption of IHO1-HORMAD1 complex and/or ANKRD31. G Model for the assembly of DSB-factor clusters on axis. Black arrows represent promotion of (i) IHO1 phosphorylation and (ii) seeding or (iii) growth of DSB-factor clusters by CDC7-DBF4, IHO1 (in particular, phosphorylated IHO1 C-terminus) and ANKRD31, respectively. See also related Supplementary Fig. and and Supplementary Table . Source data are provided as a Source Data file.

Journal: Nature Communications

Article Title: Seeding the meiotic DNA break machinery and initiating recombination on chromosome axes

doi: 10.1038/s41467-024-47020-1

Figure Lengend Snippet: A, B, D Numbers of small MEI4-REC114 co-clusters ( A ), MEI4 intensities in MEI4-REC114 co-clusters ( B , data points show median cluster intensities per cell), and DMC1 focus numbers ( D ) in spermatocytes of adult mice. Zygo-pachytene ( D) , only in SC-defective backgrounds) is equivalent to a mix of late-zygotene and early pachytene stages which are indistinguishable if SC is defective. Pooled data is shown from 6 ( A, B ) or 2 ( D ) mice of each genotype. Bars are medians, n=cell numbers. Two-tailed Mann Whitney U-Test, ns= P > 0.05, *= P < 0.05, **=P < 0.01, ***= P < 0.001, ****= P < 0.0001. Exact P values: ( A ), wild type (wt) versus Ankrd31 −/− pre-leptotene P = 5.35e-13, early zygotene P = 0.0001596, all the others P < 2.2e-16, ( B ), all comparisons in pre-leptotene and leptotene P < 2.2e-16, early zygotene, wt vs. Ankrd31 −/− P = 0.3047, Ankrd31 −/− vs. Iho1 C7Δ/C7Δ P = 0.001186, Ankrd31 −/− vs. Hormad1 −/− P = 7.19e-5, ( D ), leptotene, wt vs. Ankrd31 −/− P = 1.07e-6, Ankrd31 −/− vs. Iho1 C7Δ/C7Δ P = 0.01621, Iho1 C7Δ/C7Δ vs. Ankrd31 −/− Iho1 C7Δ/C7Δ P = 5.28e-5, Ankrd31 −/− vs. Hormad1 −/− P = 0.1945, Hormad1 −/− vs. Ankrd31 −/− Hormad1 −/− P = 0.175, early zygotene, wt vs. Ankrd31 −/− P = 5.82e-10, Ankrd31 −/− vs. Iho1 C7Δ/C7Δ P = 0.00041, Iho1 C7Δ/C7Δ vs. Ankrd31 −/− Iho1 C7Δ/C7Δ P = 1.47e-13, Ankrd31 −/− vs. Hormad1 −/− P = 0.0002882, Hormad1 −/− vs. Ankrd31 −/− Hormad1 −/− P < 2.2e-16, late zygotene, wt vs. Ankrd31 −/− P = 0.0004372, Ankrd31 −/− vs. Iho1 C7Δ/C7Δ P = 1.48e-13, Iho1 C7Δ/C7Δ vs. Ankrd31 −/− Iho1 C7Δ/C7Δ P = 5.35e-16, Ankrd31 −/− vs. Hormad1 −/− P < 2.2e-16, Hormad1 −/− vs. Ankrd31 −/− Hormad1 −/− P = 2.49e-11, early pachytene, wt vs. Ankrd31 −/− P = 0.0001234, Ankrd31 −/− vs. Iho1 C7Δ/C7Δ P = 1.1e-10. C Immunostaining in leptotene spermatocytes from adult mice. Bars, 10 µm. E Radiograph of immunoprecipitated and radioactively labeled SPO11-oligo complexes from testes of adult mice. Bar, SPO11-specific signal, asterisk, nonspecific labelling, and arrowhead, immunoglobulin heavy-chain. Radioactive signals were background-corrected ( Iho1 −/− , signal=0) and normalized to wild-type control (1). Means and standard deviations are from n = 2 biological replicates. F Schematic summary of phenotypes caused by the disruption of IHO1-HORMAD1 complex and/or ANKRD31. G Model for the assembly of DSB-factor clusters on axis. Black arrows represent promotion of (i) IHO1 phosphorylation and (ii) seeding or (iii) growth of DSB-factor clusters by CDC7-DBF4, IHO1 (in particular, phosphorylated IHO1 C-terminus) and ANKRD31, respectively. See also related Supplementary Fig. and and Supplementary Table . Source data are provided as a Source Data file.

Techniques: Two Tailed Test, MANN-WHITNEY, Immunostaining, Immunoprecipitation, Labeling, Disruption

Review

Structured Review

Images

1) Product Images from "Ectopically Expressed Meiosis-Specific Cancer Testis Antigen HORMAD1 Promotes Genomic Instability in Squamous Cell Carcinomas."

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