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).

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, 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).

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: 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).

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, 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);

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: 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.

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

Journal: PLoS Genetics

Article Title: Phosphorylation of Chromosome Core Components May Serve as Axis Marks for the Status of Chromosomal Events during Mammalian Meiosis

doi: 10.1371/journal.pgen.1002485

Figure Lengend Snippet: HORMAD1 is phosphorylated at Ser 375 on unsynapsed chromosomes.

Article Snippet: The following antibodies were also used: guinea pig anti-SYCP2, anti-SMC1β, anti-STAG3, anti-REC8 and anti-SYCP1 antibodies ; guinea pig anti-HORMAD1antibody ; rabbit anti-HORMAD1 antibody (13917-1-AP) from Proteintech Group; rabbit anti-pS/T-Q antibody (#2851) from Cell Signaling Technology; rabbit anti-pS1083 antibodies (A300-480A and IHC-00070) from Bethyl Laboratories; mouse and rabbit anti-γH2AX antibodies (#05-636 and #07-164) from Millipore; mouse anti-SYCP3 (sc-74569), rabbit anti-HORMAD2 (sc-82192), goat anti-SMC3 (sc-8198) and goat anti-ATR (sc-1887) antibodies from Santa Cruz Biotechnology; rabbit anti-SMC3 (ab9263) and rabbit anti-SYCP1 (ab15090) antibodies from Abcam; mouse anti-SYCP1 antibody (a gift from C. Heyting).

Techniques:

Journal: PLoS Genetics

Article Title: Phosphorylation of Chromosome Core Components May Serve as Axis Marks for the Status of Chromosomal Events during Mammalian Meiosis

doi: 10.1371/journal.pgen.1002485

Figure Lengend Snippet: (A) Testis nuclear extracts were immunoprecipitated with the anti-SMC3 antibody, followed by treatment with (+) or without (−) phosphatase (PPase) and phosphatase inhibitors (Inhibitor). 80% of the immunoprecipitated SMC3 and the rest were separated on a gradient gel and immunoblotted with antibodies against the Ser 1083 -phosphorylated form of SMC3 (pS1083) and normal SMC3, respectively. (B) Testis nuclear extracts were immunoprecipitated without (Mock) or with the anti-pS1083 antibody. The immunoprecipitates were probed with antibodies against meiotic chromosome axis components. The asterisk marks a non-specific band. (C) Nuclear spreads of spermatocytes were labeled with anti-pS1083, anti-SYCP3 and anti-HORMAD1 antibodies. Arrowheads indicate the XY bivalent. Bars, 10 µm.

Article Snippet: The following antibodies were also used: guinea pig anti-SYCP2, anti-SMC1β, anti-STAG3, anti-REC8 and anti-SYCP1 antibodies ; guinea pig anti-HORMAD1antibody ; rabbit anti-HORMAD1 antibody (13917-1-AP) from Proteintech Group; rabbit anti-pS/T-Q antibody (#2851) from Cell Signaling Technology; rabbit anti-pS1083 antibodies (A300-480A and IHC-00070) from Bethyl Laboratories; mouse and rabbit anti-γH2AX antibodies (#05-636 and #07-164) from Millipore; mouse anti-SYCP3 (sc-74569), rabbit anti-HORMAD2 (sc-82192), goat anti-SMC3 (sc-8198) and goat anti-ATR (sc-1887) antibodies from Santa Cruz Biotechnology; rabbit anti-SMC3 (ab9263) and rabbit anti-SYCP1 (ab15090) antibodies from Abcam; mouse anti-SYCP1 antibody (a gift from C. Heyting).

Techniques: Immunoprecipitation, Labeling

Journal: PLoS Genetics

Article Title: Phosphorylation of Chromosome Core Components May Serve as Axis Marks for the Status of Chromosomal Events during Mammalian Meiosis

doi: 10.1371/journal.pgen.1002485

Figure Lengend Snippet: (A and D) The insoluble fraction of testis nuclear extracts was prepared from Atm −/− (A) and Brca1 Δ11/Δ11 Trp53 +/− ( Brca1 Δ ) (D) males and probed with antibodies against meiotic chromosome axis components. (B and E) Testis nuclear extracts from Atm −/− (B) and Brca1 Δ11/Δ11 Trp53 +/− (E) males were immunoprecipitated with the anti-HORMAD1 antibody. 80% of the immunoprecipitated HORMAD1 and the rest were separated on a gradient gel and immunoblotted with anti-pS375 and anti-HORMAD1 antibodies, respectively. The asterisk marks a non-specific band. (C and F) Testis nuclear extracts from Atm −/− (C) and Brca1 Δ11/Δ11 Trp53 +/− (F) males were immunoprecipitated with the anti-SMC3 antibody. 80% of the immunoprecipitated SMC3 and the rest were separated on a gradient gel and immunoblotted with anti-pS1083 and anti-SMC3 antibodies, respectively. (G) Nuclear spreads of Brca1 Δ11/Δ11 Trp53 +/− pachytene spermatocytes were labeled with anti-pS375, anti-SYCP3 and anti-SYCP1 antibodies. Arrowheads indicate the XY bivalent. Bar, 10 µm.

Article Snippet: The following antibodies were also used: guinea pig anti-SYCP2, anti-SMC1β, anti-STAG3, anti-REC8 and anti-SYCP1 antibodies ; guinea pig anti-HORMAD1antibody ; rabbit anti-HORMAD1 antibody (13917-1-AP) from Proteintech Group; rabbit anti-pS/T-Q antibody (#2851) from Cell Signaling Technology; rabbit anti-pS1083 antibodies (A300-480A and IHC-00070) from Bethyl Laboratories; mouse and rabbit anti-γH2AX antibodies (#05-636 and #07-164) from Millipore; mouse anti-SYCP3 (sc-74569), rabbit anti-HORMAD2 (sc-82192), goat anti-SMC3 (sc-8198) and goat anti-ATR (sc-1887) antibodies from Santa Cruz Biotechnology; rabbit anti-SMC3 (ab9263) and rabbit anti-SYCP1 (ab15090) antibodies from Abcam; mouse anti-SYCP1 antibody (a gift from C. Heyting).

Techniques: Immunoprecipitation, Labeling

Journal: PLoS Genetics

Article Title: Phosphorylation of Chromosome Core Components May Serve as Axis Marks for the Status of Chromosomal Events during Mammalian Meiosis

doi: 10.1371/journal.pgen.1002485

Figure Lengend Snippet: (A) The insoluble fraction of testis nuclear extracts was probed with antibodies against meiotic chromosome axis components. (B) The Ser 375 -phosphorylated form of HORMAD1 was examined as in . (C) The Ser 1083 -phosphorylated form of SMC3 was examined as in . (D) Nuclear spreads of Spo11 −/− zygotene-like spermatocytes were labeled with anti-pS375, anti-SYCP3 and anti-HORMAD1 antibodies. (E) Nuclear spreads of Spo11 −/− zygotene-like spermatocytes were labeled with anti-pS1083, anti-SYCP3 and anti-HORMAD1 antibodies. Bars, 10 µm.

Article Snippet: The following antibodies were also used: guinea pig anti-SYCP2, anti-SMC1β, anti-STAG3, anti-REC8 and anti-SYCP1 antibodies ; guinea pig anti-HORMAD1antibody ; rabbit anti-HORMAD1 antibody (13917-1-AP) from Proteintech Group; rabbit anti-pS/T-Q antibody (#2851) from Cell Signaling Technology; rabbit anti-pS1083 antibodies (A300-480A and IHC-00070) from Bethyl Laboratories; mouse and rabbit anti-γH2AX antibodies (#05-636 and #07-164) from Millipore; mouse anti-SYCP3 (sc-74569), rabbit anti-HORMAD2 (sc-82192), goat anti-SMC3 (sc-8198) and goat anti-ATR (sc-1887) antibodies from Santa Cruz Biotechnology; rabbit anti-SMC3 (ab9263) and rabbit anti-SYCP1 (ab15090) antibodies from Abcam; mouse anti-SYCP1 antibody (a gift from C. Heyting).

Techniques: Labeling

Journal: PLoS Genetics

Article Title: Phosphorylation of Chromosome Core Components May Serve as Axis Marks for the Status of Chromosomal Events during Mammalian Meiosis

doi: 10.1371/journal.pgen.1002485

Figure Lengend Snippet: (A) The insoluble fraction of testis nuclear extracts was probed with antibodies against meiotic chromosome axis components. (B) The Ser 375 -phosphorylated form of HORMAD1 was examined as in . (C) The Ser 1083 -phosphorylated form of SMC3 was examined as in . (D) Nuclear spreads of Sycp3 −/− zygotene-like spermatocytes were labeled with anti-pS375, anti-SYCP1 and anti-HORMAD1 antibodies. (E) Nuclear spreads of Sycp3 −/− zygotene-like spermatocytes were labeled with anti-pS1083, anti-SYCP1 and anti-HORMAD1 antibodies. Bars, 10 µm.

Article Snippet: The following antibodies were also used: guinea pig anti-SYCP2, anti-SMC1β, anti-STAG3, anti-REC8 and anti-SYCP1 antibodies ; guinea pig anti-HORMAD1antibody ; rabbit anti-HORMAD1 antibody (13917-1-AP) from Proteintech Group; rabbit anti-pS/T-Q antibody (#2851) from Cell Signaling Technology; rabbit anti-pS1083 antibodies (A300-480A and IHC-00070) from Bethyl Laboratories; mouse and rabbit anti-γH2AX antibodies (#05-636 and #07-164) from Millipore; mouse anti-SYCP3 (sc-74569), rabbit anti-HORMAD2 (sc-82192), goat anti-SMC3 (sc-8198) and goat anti-ATR (sc-1887) antibodies from Santa Cruz Biotechnology; rabbit anti-SMC3 (ab9263) and rabbit anti-SYCP1 (ab15090) antibodies from Abcam; mouse anti-SYCP1 antibody (a gift from C. Heyting).

Techniques: Labeling

Journal: PLoS Genetics

Article Title: Phosphorylation of Chromosome Core Components May Serve as Axis Marks for the Status of Chromosomal Events during Mammalian Meiosis

doi: 10.1371/journal.pgen.1002485

Figure Lengend Snippet: (A–C) Nuclear spreads of wild-type (A), Sycp3 −/− (B) and Spo11 −/− (C) zygotene-like spermatocytes were labeled with anti-γH2AX, anti-HORMAD1 and anti-SYCP1 antibodies. (D–G) Nuclear spreads of wild-type (D), Sycp3 −/− (E), Sycp1 −/− (F) and Tex12 −/− (G) zygotene-like spermatocytes were labeled with anti-γH2AX, anti-REC8 and anti-ATR antibodies. Arrowheads indicate the position of the pseudo-sex body-like staining of γH2AX. Bars, 10 µm.

Article Snippet: The following antibodies were also used: guinea pig anti-SYCP2, anti-SMC1β, anti-STAG3, anti-REC8 and anti-SYCP1 antibodies ; guinea pig anti-HORMAD1antibody ; rabbit anti-HORMAD1 antibody (13917-1-AP) from Proteintech Group; rabbit anti-pS/T-Q antibody (#2851) from Cell Signaling Technology; rabbit anti-pS1083 antibodies (A300-480A and IHC-00070) from Bethyl Laboratories; mouse and rabbit anti-γH2AX antibodies (#05-636 and #07-164) from Millipore; mouse anti-SYCP3 (sc-74569), rabbit anti-HORMAD2 (sc-82192), goat anti-SMC3 (sc-8198) and goat anti-ATR (sc-1887) antibodies from Santa Cruz Biotechnology; rabbit anti-SMC3 (ab9263) and rabbit anti-SYCP1 (ab15090) antibodies from Abcam; mouse anti-SYCP1 antibody (a gift from C. Heyting).

Techniques: Labeling, Staining

Journal: PLoS Genetics

Article Title: Phosphorylation of Chromosome Core Components May Serve as Axis Marks for the Status of Chromosomal Events during Mammalian Meiosis

doi: 10.1371/journal.pgen.1002485

Figure Lengend Snippet: (A) Schematic representation of the model for regulation of phosphorylation of meiotic chromosomal proteins at S/T-Q motifs. In response to SPO11-formed DSBs (arrow 1), ATM phosphorylates histone H2AX (arrow 2) and ATR phosphorylates HORMAD1/2 (arrow 3) and SMC3 (arrow 4). Phosphorylated HORMAD1/2 serves as a marker for unsynapsis and contributes to the correct localization of ATR at unsynapsed chromosomal regions (arrow 5). At the unsynapsed chromosomes, ATR phosphorylates H2AX to promote MSUC (arrow 6), as well as HORMAD1/2 (arrow 7) and SMC3 (arrow 8). Phosphorylated HORMAD1/2 further stabilizes ATR (arrow 9) at unsynapsed chromosomes and ATR further phosphorylates HORMAD1/2 (arrow 10), amplifying the unsynapsis signal via the positive feedback loop (arrow 9 and 10). (B) The status of chromosome synapsis can be indicated by the presence or absence of HORMAD1/2 and phosphorylation of HORMAD1 and SMC3. At unsynapsed chromosomal regions, the chromosome axis contains the S/T-Q motif-phosphorylated forms of HORMAD1/2 and SMC3. When homologs are synapsed, HORMAD1/2 and the Ser 1083 -phosphorylated form of SMC3 are displaced from the chromosome axis. After desynapsis, HORMAD1/2 is again included in the chromosome axis but HORMAD1 (and possibly HORMAD2) is not phosphorylated at the S/T-Q motif. Distribution of the phosphorylated forms of other components of the chromosome axis remains to be determined.

Article Snippet: The following antibodies were also used: guinea pig anti-SYCP2, anti-SMC1β, anti-STAG3, anti-REC8 and anti-SYCP1 antibodies ; guinea pig anti-HORMAD1antibody ; rabbit anti-HORMAD1 antibody (13917-1-AP) from Proteintech Group; rabbit anti-pS/T-Q antibody (#2851) from Cell Signaling Technology; rabbit anti-pS1083 antibodies (A300-480A and IHC-00070) from Bethyl Laboratories; mouse and rabbit anti-γH2AX antibodies (#05-636 and #07-164) from Millipore; mouse anti-SYCP3 (sc-74569), rabbit anti-HORMAD2 (sc-82192), goat anti-SMC3 (sc-8198) and goat anti-ATR (sc-1887) antibodies from Santa Cruz Biotechnology; rabbit anti-SMC3 (ab9263) and rabbit anti-SYCP1 (ab15090) antibodies from Abcam; mouse anti-SYCP1 antibody (a gift from C. Heyting).

Techniques: Phospho-proteomics, Marker

Journal: Molecular cell

Article Title: Impeding DNA Break Repair Enables Oocyte Quality Control

doi: 10.1016/j.molcel.2018.08.031

Figure Lengend Snippet: KEY RESOURCE TABLE

Article Snippet: Antibodies mouse anti-p63 Santa Cruz Biotechnology sc-25268 rabbit anti-MVH Abcam ab13840 rabbit anti-SYCP3 Santa Cruz Biotechnology sc-33195 anti-rabbit 488 Molecular Probes A11070 anti-mouse 594 Molecular Probes A11020 anti-rabbit 568 Molecular Probes A11036 anti-mouse 488 Molecular Probes A11029 Biotin-conjugated anti-mouse secondary antibody ThermoFisher Scientific A16076 human anti-centromere antibodies ImmunoVision ACA; HCT-0100 anti-human 555 secondary antibody Molecular Probes A-21433 mouse anti-SYCP3 Santa Cruz Biotechnology sc-74568 guinea pig anti-SYCP2 a gift from Christer Höög N/A rabbit monoclonal anti-RPA32 Abcam ab76420 rabbit anti-REC8 a gift from Scott Keeney N/A mouse monoclonal anti-γH2AX Millipore 05-636 rabbit polyclonal anti-γH2AX Bethyl laboratories A300-081A goat polyclonal anti-Caspase3 (L-18) Santa Cruz Biotechnology Sc-1225 rabbit anti-HORMAD1 a gift from Attila Toth N/A anti-mouse 555 Molecular Probes A21425 anti-guinea pig fluorescein isothiocyanate Jackson Labs 106-096-006 FITC Chemicals, Peptides, and Recombinant Proteins Protein Block, Serum Free, Blocking, Unconjugated, Liquid form, Immunohistochemistry, 110 mL Agilent Dako X090930-2 ABC-DAB Peroxidase system Vector Laboratories Cat# PK-6100 Tyrode’s Solution, Acidic Sigma-Aldrich Cat# T1788 milrnone Sigma-Aldrich Cat# 78415-72-2 3% BSA Thermo Fisher Scientific Cat# AM2616 ProLong Gold antifade reagent (Molecular Probes) Thermo Fisher Scientific Cat# P36930 M2 medium Sigma-Aldrich Cat# M7167 Open in a separate window KEY RESOURCE TABLE REAGENT or RESOURCE SOURCE IDENTIFIER Software and Algorithms Volocity 6.3 Perkin Elmer N/A Aperio ImageScope software Leica N/A Open in a separate window Highlights RNF212, a SUMO ligase required for crossovers, also mediates oocyte quality control RNF212 promotes apoptosis of oocytes that experienced defects in meiotic prophase I RNF212 works with HORMAD1 to impede repair of residual DSBs as chromosomes desynapse Impeded DSBs signal that meiosis was defective and trigger oocyte apoptosis

Techniques: Recombinant, Blocking Assay, Immunohistochemistry, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: The cancer testes antigen, HORMAD1, limits genomic instability in cancer cells by protecting stalled replication forks

doi: 10.1016/j.jbc.2023.105348

Figure Lengend Snippet: HORMAD1 protects stalled replication forks from degradation . A , H1395 cells were labeled with 20 μM EdU for 10 min, followed by fixation and SIRF. Each dot represents the number of HORMAD1-EDU foci per nuclei. Mean is indicated. Left : images of SIRF signal in individual nuclei. The scale bar represents 10 μm. B , DNA fiber assay in H1395 cells according to the schematic ( top ). Left : each circle represents the length of individual tracts with median indicated. p value calculated by Mann–Whitney test; n = 3 . Right : representative image of a single DNA fiber. The scale bar represents 10 μm. C , DNA fiber assay in H1395 cells according to the schematic ( top ). (i) Bars represent the mean of forks with only IdU labeling as a percentage of all forks observed (error bars represent SEM). p value calculated by an unpaired t test; n = 3. (ii) Bars represent the mean of tracts labeled with both IdU and CldU as a percentage of total tracts observed (error bars represent SEM). p value calculated by an unpaired t test; n = 3. (iii) Graph represents the IdU length of individual fibers. Median is indicated. p value calculated by Mann-Whitney test; n = 3. Right : representative images of fibers are shown. The scale bar represents 10 μm. D , H1395 cells were labeled and treated as indicated in schematic ( top ) followed by a DNA fiber assay. Each dot represents the ratio of CldU length to IdU length for an individual tract. Median is indicated. p value was calculated by Mann–Whitney test; n =3. Below : Representative images of fibers are shown, the scale bar represents 10 μm. E , top : As in ( D ). Bottom : Parallel lysates were obtained, and an immunoblot performed with indicated antibodies. F , H1395 cells were labeled and treated as indicated in schematic ( top ) followed by a DNA fiber assay. Each dot represents the ratio of CldU length to IdU length for an individual tract. Median is indicated. p value was calculated by Mann–Whitney test. n = 2, where at least 100 fibers were quantified for each biological replicate. G , H1395 cells were labeled and treated as indicated in schematic ( top ) followed by a DNA fiber assay. Each dot represents the ratio of CldU length to IdU length for an individual tract. Median is indicated. p value was calculated by Mann–Whitney test. n = 2, where at least 100 fibers were quantified for each biological replicate. H , HORMAD1 was depleted for 72 h in indicated LUAD cell lines. Cells were labeled and treated according to the schematic ( top ). Points represent the CldU/IdU ratio for individual tracts. Median is indicated. p value calculated by Mann–Whitney test; n = 3. Below : parallel lysates were obtained, and an immunoblot performed for indicated antibodies. 50kDA marker is indicated on each HORMAD1 blot. CldU, 5-chloro-2′-deoxyuridine; IdU, 5-iodo-2′-deoxyuridine;LUAD, lung adenocarincoma.

Article Snippet: Cells were incubated with primary antibodies overnight at 4 °C (mouse anti-biotin antibody (1:250, B7653, Sigma-Aldrich) with rabbit anti-HORMAD1 antibody (HPA037850, Sigma-Aldrich)).

Techniques: Labeling, MANN-WHITNEY, Western Blot, Marker

Journal: The Journal of Biological Chemistry

Article Title: The cancer testes antigen, HORMAD1, limits genomic instability in cancer cells by protecting stalled replication forks

doi: 10.1016/j.jbc.2023.105348

Figure Lengend Snippet: HORMAD1 protects the replication fork from nascent DNA degradation by the MRE11-DNA2-BLM enzymatic axis . A , H1395 cells were transfected with indicated siRNAs for 72 h before labeling according to the schematic ( top ) and a DNA fiber assay. Graph represents the CldU/IdU ratio for DNA fibers with the median indicated. p value calculated by Mann-Whitney t test; n = 3. Below : Parallel lysates were obtained and immunoblotted with indicated antibodies. B , H1395 cells were transfected with indicated siRNAs for 72 h followed by treatment according to the schematic ( top ). p value calculated by Mann–Whitney t test; n = 3. Graph as in A . C and D , as in A . E , H1395 cells were transfected with indicated siRNAs for 72 h. Cells were exposed to DMSO or C5 for 2 h prior to labeling ( top ) and DNA fiber assay. Graph represents the CldU/IdU ratio for individual fibers with median indicated. p value calculated by Mann–Whitney t test; n ≥ 3. F , as in A . CldU, 5-chloro-2′-deoxyuridine; DMSO, dimethylsulfoxide;IdU, 5-iodo-2′-deoxyuridine.

Article Snippet: Cells were incubated with primary antibodies overnight at 4 °C (mouse anti-biotin antibody (1:250, B7653, Sigma-Aldrich) with rabbit anti-HORMAD1 antibody (HPA037850, Sigma-Aldrich)).

Techniques: Transfection, Labeling, MANN-WHITNEY

Journal: The Journal of Biological Chemistry

Article Title: The cancer testes antigen, HORMAD1, limits genomic instability in cancer cells by protecting stalled replication forks

doi: 10.1016/j.jbc.2023.105348

Figure Lengend Snippet: HORMAD1 promotes recruitment of RAD51 to stalled replication forks . A , H1395 cells were labeled as indicated in the diagram ( top ). Control cells were fixed following EdU labeling. Immunofluorescence was performed for IdU and EdU under nondenaturing conditions. Graph represents IdU nuclear fluorescence intensity in EdU positive cells. Mean is indicated. p value calculated by Mann–Whitney test; n = 3. Right panel : representative images of cells are shown. The scale bar represents 10 μm. B , indicated H1395 cells were labeled with EdU then exposed to HU or stained for RPA and EdU. Box plot was derived by measuring the mean RPA foci in EdU positive cells. p value calculated by Mann–Whitney test; n = 3. Right panel : representative images of cells are shown. The scale bar represents 10 μm. C , As in ( B ), except cells were stained for RAD51. Right panel : representative images of cells are shown. D , endogenous HORMAD1 was coimmunoprecipitated in H1395 cells and lysate samples were immunoblotted for HORMAD1 and WRNIP1. For loading, 5% of the input was taken from lysates and an IgG rabbit antibody pull-down was used as a control. Representative blot of n = 2. E , H1395 cells were transfected with indicated siRNAs for 72 h before labeling according to the schematic ( top ) and a DNA fiber assay. Graph represents the CldU/IdU ratio for DNA fibers with the median indicated. p value calculated by Mann–Whitney t test; n = 3. Below : Parallel lysates were obtained and immunoblotted with indicated antibodies. ICldU, 5-chloro-2′-deoxyuridine; dU, 5-iodo-2′-deoxyuridine; EdU, 5-ethynyl-2′-deoxyuridine; HU, hydroxyurea.

Article Snippet: Cells were incubated with primary antibodies overnight at 4 °C (mouse anti-biotin antibody (1:250, B7653, Sigma-Aldrich) with rabbit anti-HORMAD1 antibody (HPA037850, Sigma-Aldrich)).

Techniques: Labeling, Immunofluorescence, Fluorescence, MANN-WHITNEY, Staining, Derivative Assay, Transfection

Journal: The Journal of Biological Chemistry

Article Title: The cancer testes antigen, HORMAD1, limits genomic instability in cancer cells by protecting stalled replication forks

doi: 10.1016/j.jbc.2023.105348

Figure Lengend Snippet: HORMAD1 mitigates DNA damage during DNA replication stress . A , indicated H1395 cells were labeled with EdU for 10 min then exposed to HU or fixed. Immunofluorescence was performed for mean γH2AX and EdU. Center line indicates the median, bounds of box indicate the first and third quartile, and whiskers indicate the 10th and 90th percentile for mean γH2AX nuclear fluorescence intensity in EdU positive cells. The scale bar represents 10 μm; p value calculated by Mann–Whitney test; n ≥ 3. Right panel : representative images of cells are shown. B , indicated H1395 cells were labeled with BrdU for 20 min and processed immediately or exposed to HU and then processed for an alkaline comet assay. Left : Box plot was derived from measuring the tail moment in BrdU positive cells and boundaries are indicated as in ( A ). p value calculated by Mann-Whitney test; n = 3. Right : representative images of comets are shown. The scale bar represents 50 μm. C , indicated H1395 cells were processed immediately or exposed to either HU or 1 μm Camptothecin and processed for a neutral comet assay. Left : Box plot was derived from measuring the tail moment in all cells and boundaries are indicated as in ( A ). p value calculated by Mann–Whitney test; n = 2. D , H1395 cells were treated as indicated in schematic ( top ) prior to performing a metaphase spread assay. Each data point represents one metaphase spread. Mean is indicated. Error bars represent ± SEM. Right : Representative pictures magnified from 100×. The scale bar represents 10 μm. Inset scale bar represents 2.5 μm. p value calculated by Mann-Whitney test; n = 3 experiments, with a minimum of 30 metaphases per condition per experiment. E , schematic indicating tumor evolution and replication stress mitigation by HORMAD1. Created with BioRender.com . BrdU, bromodeoxyuridine; EdU, 5-ethynyl-2′-deoxyuridine; HU, hydroxyurea.

Article Snippet: Cells were incubated with primary antibodies overnight at 4 °C (mouse anti-biotin antibody (1:250, B7653, Sigma-Aldrich) with rabbit anti-HORMAD1 antibody (HPA037850, Sigma-Aldrich)).

Techniques: Labeling, Immunofluorescence, Fluorescence, MANN-WHITNEY, Alkaline Single Cell Gel Electrophoresis, Derivative Assay, Neutral Comet Assay

Journal: Cell Proliferation

Article Title: ZFP541 and KCTD19 regulate chromatin organization and transcription programs for male meiotic progression

doi: 10.1111/cpr.13567

Figure Lengend Snippet: Zfp541 −/− spermatocytes show aberrant chromatin organization. (A–C) The detached chromosome ends in Zfp541 KO pachytene spermatocytes. (A) Immunostaining to visualize telomere binding TRF1. (B) FISH with telomeric repeat DNA probe to visualize telomeres. (C) Immunostaining with anti‐centromere antibody ACA to show centromeres. (D, E) Representative images to show HORMAD1 (D) and TRIP13 (E) in spread spermatocytes.

Article Snippet: Primary antibodies used for immunostaining were as follows: mouse anti‐SYCP3 (1:500; Abcam, ab97672), rabbit anti‐SYCP3 (1:500; Abcam, ab15093), rabbit anti‐SYCP1 (1:500; Abcam, ab15090), guinea pig anti‐ZFP541 (1:100), mouse anti‐γH2AX (Ser139) (1:300; Millipore, 05‐636), mouse anti‐MLH1 (1:100; BD Biosciences, 550838), rabbit anti‐phospho‐histone H3 (Ser10) (1:200; Millipore, 06‐570), mouse anti‐TRF1 (1:200; Abcam, ab10579), human anti‐ACA (1:300; antibodies incorporated, 15‐234‐0001), telomere Probe (5 nM; PNA Bio, F1009), rabbit anti‐TRIP13 (1:200; Proteintech, 19602‐1‐AP), rabbit anti‐HORMAD1 (1:200; ABclonal, WG‐04467D), mouse anti‐CDK2 (1:100; Santa Cruz Biotechnology, SC‐6248), rabbit anti‐RPA2 (1:200; Abcam, ab76420), rabbit anti‐RAD51 (1:200; Thermo Fisher Scientific, PA5‐27195), rabbit anti‐DMC1 (1:200; Proteintech, 13714‐1‐AP50), rabbit anti‐TEX11 (1:200), rabbit anti‐MSH4 (1:200; Abcam, ab58666), rabbit anti‐RNF212 (1:200; a gift from Mengcheng Luo, Wuhan University), guinea pig anti‐HEI10 (1:150), guinea pig anti‐H1t (1:200), rabbit anti‐SUMO1 (1:200; Cell Signalling Technology, #4930), rabbit anti‐SUMO2/3 (1:200; Cell Signalling Technology, #4971), rabbit anti‐Ubiquitin (1:200; Ubiquigent, 68‐0121‐500), mouse anti‐20S proteasome β5(1:50; Santa Cruz A‐10 sc:393931), rabbit anti‐Histone H4 (1:200; Abcam, ab10807).

Techniques: Immunostaining, Binding Assay

Journal: Science Advances

Article Title: SKP1 drives the prophase I to metaphase I transition during male meiosis

doi: 10.1126/sciadv.aaz2129

Figure Lengend Snippet: ( A ) Western blot analysis of SKP1 in developing mouse testes. Timing of the first appearance of spermatogonium, pachytene spermatocytes, round spermatids (spt), and elongated spermatids in developing testes is shown. SYCP3, meiosis-specific protein control; ACTB, loading control. ( B ) Spatiotemporal expression pattern of SKP1 in postnatal male germ cells. DAPI, 4′,6-diamidino-2-phenylindole. ( C ) Localization of SKP1 to synapsed regions of the SC (indicated by arrows) in wild-type (WT) spermatocytes. ( D ) Super-resolution localization of SKP1 and SYCP3 in pachytene spermatocytes. ( E ) Super-resolution localization of SKP1 and SYCP1 in pachytene spermatocytes. SYCP1 is a component of both CE and transverse filaments. ( F ) Localization of SKP1 to the SC between sister chromatids in Rec8 −/− spermatocytes. ( G ) Super-resolution localization of SKP1 in Rec8 −/− spermatocytes. ( H ) Super-resolution localization of HORMAD1 in Rec8 −/− spermatocytes. Scale bars, 10 μm.

Article Snippet: The following primary antibodies were used for immunofluorescence analyses: rabbit anti-SYCP1 (1:100, catalog number ab15090, Abcam), mouse anti-SYCP1 (1:200; a gift from C. Hoog), guinea pig anti-SYCP2 , mouse anti-SYCP3 (1:200; catalog number ab97672, Abcam), rabbit anti-SYCP3 (1:200; 23024-1-AP), rabbit anti-SKP1 (1:50; catalog number ab10546, Abcam), rabbit anti-HORMAD1 , rabbit anti-HORMAD1 (1:200; 13917-1-AP, Proteintech Group), rabbit anti-HORMAD2 , mouse anti-γH2AX (1:500; catalog number 16-202A, clone JBW301, Millipore), guinea pig anti-H1T (1:500; a gift from M. A. Handel) , rabbit anti-RPA2 (1:200; UP2436) , mouse anti-MLH1 (1:50; catalog number 550838, clone G168-15, BD Biosciences), human anti-CREST (1:100; 15-234, Antibodies Incorporated), mouse anti-CCNB1 (1:200; ab72, Abcam), rabbit anti-pHH3 (1:300; 9701S, Cell Signaling Technology), rabbit anti-PLK1 (1:50; UP2456; this study), mouse anti-BUB1 (1:50; a gift from Y. Watanabe) ( , ), and rabbit anti–CENP-C (1:500; a gift from Y. Watanabe) ( ).

Techniques: Western Blot, Expressing

Journal: Science Advances

Article Title: SKP1 drives the prophase I to metaphase I transition during male meiosis

doi: 10.1126/sciadv.aaz2129

Figure Lengend Snippet: ( A ) Localization of HORMAD1 in pachynema and diplonema. The plot shows the count of HORMAD1-high versus HORMAD1-low Skp1 cKO diplonema. ( B ) Localization of HORMAD2 in pachynema and diplonema. The count of HORMAD2-high versus HORMAD2-low Skp1 cKO diplonema is shown in the plot. ( C ) Super-resolution localization of HORMAD1 to the SC LEs in Skp1 cKO pachynema. Scale bars, 10 μm. ( D ) Western blot analysis of SKP1, HORMAD1, HORMAD2, TRIP13, CUL1, and SYCP3 in Skp1 cKO testes at 2, 4, 6, 16, and 32 dpt.

Article Snippet: The following primary antibodies were used for immunofluorescence analyses: rabbit anti-SYCP1 (1:100, catalog number ab15090, Abcam), mouse anti-SYCP1 (1:200; a gift from C. Hoog), guinea pig anti-SYCP2 , mouse anti-SYCP3 (1:200; catalog number ab97672, Abcam), rabbit anti-SYCP3 (1:200; 23024-1-AP), rabbit anti-SKP1 (1:50; catalog number ab10546, Abcam), rabbit anti-HORMAD1 , rabbit anti-HORMAD1 (1:200; 13917-1-AP, Proteintech Group), rabbit anti-HORMAD2 , mouse anti-γH2AX (1:500; catalog number 16-202A, clone JBW301, Millipore), guinea pig anti-H1T (1:500; a gift from M. A. Handel) , rabbit anti-RPA2 (1:200; UP2436) , mouse anti-MLH1 (1:50; catalog number 550838, clone G168-15, BD Biosciences), human anti-CREST (1:100; 15-234, Antibodies Incorporated), mouse anti-CCNB1 (1:200; ab72, Abcam), rabbit anti-pHH3 (1:300; 9701S, Cell Signaling Technology), rabbit anti-PLK1 (1:50; UP2456; this study), mouse anti-BUB1 (1:50; a gift from Y. Watanabe) ( , ), and rabbit anti–CENP-C (1:500; a gift from Y. Watanabe) ( ).

Techniques: Western Blot

Journal: Science Advances

Article Title: SKP1 drives the prophase I to metaphase I transition during male meiosis

doi: 10.1126/sciadv.aaz2129

Figure Lengend Snippet: ( A ) Count of metaphase spermatocytes (pHH3-positive) in seminiferous tubules from WT and Skp1 cKO testes. Scale bar, 25 μm. ( B ) Western blot (WB) analysis of cell cycle regulators in Skp1 cKO testes. ( C ) Skp1 -deficient spermatocytes are incompetent for MI entry with OA induction. Scale bar, 10 μm. ( D ) Histone H1 kinase assay for the MPF activity in WT (control) and Skp1 cKO testes at 2 and 6 dpt. Immunoglobulin G (IgG) serves as a negative control. IP, immunoprecipitation. ( E ) Key functions of SKP1 during male meiosis. This diagram summarizes the major meiotic defects in Skp1 cKO testes. Note the accumulation of HORMAD1 on LEs in Y pachynema and its increased abundance on LEs in diplonema in the absence of SKP1. For illustration purpose, SKP1 and HORMAD1 are shown next to the LEs instead of overlaying.

Article Snippet: The following primary antibodies were used for immunofluorescence analyses: rabbit anti-SYCP1 (1:100, catalog number ab15090, Abcam), mouse anti-SYCP1 (1:200; a gift from C. Hoog), guinea pig anti-SYCP2 , mouse anti-SYCP3 (1:200; catalog number ab97672, Abcam), rabbit anti-SYCP3 (1:200; 23024-1-AP), rabbit anti-SKP1 (1:50; catalog number ab10546, Abcam), rabbit anti-HORMAD1 , rabbit anti-HORMAD1 (1:200; 13917-1-AP, Proteintech Group), rabbit anti-HORMAD2 , mouse anti-γH2AX (1:500; catalog number 16-202A, clone JBW301, Millipore), guinea pig anti-H1T (1:500; a gift from M. A. Handel) , rabbit anti-RPA2 (1:200; UP2436) , mouse anti-MLH1 (1:50; catalog number 550838, clone G168-15, BD Biosciences), human anti-CREST (1:100; 15-234, Antibodies Incorporated), mouse anti-CCNB1 (1:200; ab72, Abcam), rabbit anti-pHH3 (1:300; 9701S, Cell Signaling Technology), rabbit anti-PLK1 (1:50; UP2456; this study), mouse anti-BUB1 (1:50; a gift from Y. Watanabe) ( , ), and rabbit anti–CENP-C (1:500; a gift from Y. Watanabe) ( ).

Techniques: Western Blot, Kinase Assay, Activity Assay, Negative Control, Immunoprecipitation