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: Mobile DNA

Article Title: An intronic LINE-1 regulates IFNAR1 expression in human immune cells.

doi: 10.1186/s13100-023-00308-3

Figure Lengend Snippet: Fig. 4 IFNAR1.L1M2a inducibly enhances transcription of IFNAR1 during type I IFN signaling. A IFNAR1 and three nearby genes, IFNAR2, IL10RB, and IFNGR2, were all considered to be possible targets of IFNAR1.L1M2a enhancer activity due to their proximity. B RNA sequencing of three wildtype clones (gray) and four IFNAR1.L1M2a.enh knockout clones (purple) for IFNAR1.L1M2a-proximal genes during a 24 h time course of IFNβ treatment. Pairwise differential expression analysis [48] at the untreated timepoint showed slightly but significantly lower baseline expression of IFNAR1 and IFNGR2 in knockout cells, and likelihood ratio tests [48] showed significant differences in response to IFNβ treatment of IFNAR1 and IL10RB. At the 24 h timepoint, IFNAR1 expression was slightly but significantly upregulated compared to the untreated timepoint in wildtype cells but not in knockout cells, and expression of IFNAR1 was significantly lower in knockout cells. C-D Immunofluorescence was used to confirm protein-level differences in IFNAR1 expression between wildtype cells (left, gray) and knockout cells (right, purple) in untreated (top, light) and IFNβ-treated (bottom, dark) conditions. In agreement with RNA-seq data, IFNAR1 expression was induced by IFNβ treatment in wildtype cells, but not in knockout cells. Scale bar indicates 25 microns

Article Snippet: Cells were either untreated or were cultured in media containing 1000 U/ml human IFNɣ (#485-MI, R&D Systems) for 4 h before RNA extraction.

Techniques: Activity Assay, RNA Sequencing, Clone Assay, Knock-Out, Quantitative Proteomics, Expressing, Immunofluorescence

Journal: Mobile DNA

Article Title: An intronic LINE-1 regulates IFNAR1 expression in human immune cells.

doi: 10.1186/s13100-023-00308-3

Figure Lengend Snippet: Fig. 5 Deletion of IFNAR1.L1M2a.enh alters downstream transcriptional response to IFNβ. RNA sequencing of three wildtype (gray) and four IFNAR1.L1M2a.enh knockout clones (purple) during a 24 h time course of IFNβ treatment. A Likelihood Ratio Tests [48] of RNAsequencing data revealed many genes with significantly altered IFNβ responses in knockout cells compared to wildtype. Many of these genes were expected IFN stimulated genes (red) as identified by induction in wildtype cells at the 4 h timepoint [48] (Additional file 7: Table S6). Gene ontology [51] confirmed that significantly differentially responsive genes were enriched for immune-related biological processes (red). B RNA sequencing of representative expected ISGs showed a dampened initial induction in the knockout cells (purple) compared to wildtype cells (gray) and differential expression throughout the time course of IFNβ treatment according to likelihood ratio tests [48]

Article Snippet: Cells were either untreated or were cultured in media containing 1000 U/ml human IFNɣ (#485-MI, R&D Systems) for 4 h before RNA extraction.

Techniques: RNA Sequencing, Knock-Out, Clone Assay, Quantitative Proteomics