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: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Discovery of another mechanism for the inhibition of particulate guanylyl cyclases by the natriuretic peptide clearance receptor.

doi: 10.1073/pnas.2307882120

Figure Lengend Snippet: Fig. 1. Comparison of NPRA and NPRC protein expression. (A) Protein expression of NPRA and NPRC were visualized by western blotting in membrane enriched fractions prepared from a variety of mouse tissues: gonadal white adipose tissue (gWAT), inguinal white adipose tissue (iWAT), brown adipose tissue (BAT), heart, liver, kidney, and lung. (B) BAT membrane enriched fractions from wildtype and NPRC−/− mice fed either standard chow or HFD were used to visualize NPRA and NPRC protein levels. Šídák’s multiple comparisons test for two-way ANOVA indicated on graph. * P < 0.05, *** P < 0.001, **** P < 0.0001. (C) BAT protein from chow- and HFD-fed mice was separated into membrane and cytosolic enriched fractions. NPRA and NPRC were assessed by western blotting. (D) HIB-1B cells were transfected with the indicated plasmids and treated with adipocyte differentiation cocktail for 48 h. Cells were then treated with the 2.5 µM MG-132 for 16 h. Flag-NPRA and NPRC were assessed from whole-cell lysates by western blotting. Šídák’s multiple comparisons test for one-way ANOVA indicated on graph. ** P < 0.01, *** P < 0.001.

Article Snippet: For western blotting the following primary antibodies were used: Flag (Sigma #F1804), NPRA (Novus Biologicals #NBP1- 31333), NPRB (Proteintech #55113- 1- AP), NPRC (Novus Biologicals #NBP1- 31365), NanoLuc® Luciferase (R&D Systems #MAB10026), β- actin (Cell Signaling #4967).

Techniques: Comparison, Expressing, Western Blot, Membrane, Transfection

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Discovery of another mechanism for the inhibition of particulate guanylyl cyclases by the natriuretic peptide clearance receptor.

doi: 10.1073/pnas.2307882120

Figure Lengend Snippet: Fig. 2. NPRC immunoprecipitates with NPRA and NPRB. HEK 293FT cells were transfected with plasmids expressing Flag-tagged natriuretic peptide receptors. Twenty-four hours after transfection, cell lysates were immunoprecipitated with anti-Flag Affinity agarose. (A) Endogenous NPRC coimmunoprecipitated with hNPRA-Flag, hNPRB-Flag, or VEGFR1-Flag. (B) Endogeneous NPRC coimmunoprecipitated with GC-C-Flag. (C) NPRC band intensity to input of NPRC was calculated through Image J software was presented in the right panel. Šídák’s multiple comparisons test for one-way ANOVA comparing all samples to the negative control VEGFR1 for western blots in A and B are indicated on graph. *P < 0.05, ** P < 0.01, *** P < 0.001. (D) hNPRA-Flag expressing HEK 293FT cells were treated with ANP (ANP1-28, 200 nM) or a truncated form of ANP that preferentially binds NPRC (ANP4-23, 200 nM) for 30 min. Neither ANP1-28 or ANP4-23 affected the ability of endogenous NPRC to coimmunoprecipitate with hNPRA-Flag. (E) Endogenous NPRC coimmunoprecipitated with Flag-rNPRA and a truncated version of Flag- rNPRA lacking the c-terminal amino acids 528-1057 [Flag-rNPRA(1-527)].

Article Snippet: For western blotting the following primary antibodies were used: Flag (Sigma #F1804), NPRA (Novus Biologicals #NBP1- 31333), NPRB (Proteintech #55113- 1- AP), NPRC (Novus Biologicals #NBP1- 31365), NanoLuc® Luciferase (R&D Systems #MAB10026), β- actin (Cell Signaling #4967).

Techniques: Transfection, Expressing, Immunoprecipitation, Software, Negative Control, Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Discovery of another mechanism for the inhibition of particulate guanylyl cyclases by the natriuretic peptide clearance receptor.

doi: 10.1073/pnas.2307882120

Figure Lengend Snippet: Fig. 3. Endogenous natriuretic peptide receptor expression in cell models used. Expression of NPRA, NPRB, and NPRC were detected by western blot in HeLa, HEK293FT, and HAP-1 cells. β-actin was used as the loading control.

Article Snippet: For western blotting the following primary antibodies were used: Flag (Sigma #F1804), NPRA (Novus Biologicals #NBP1- 31333), NPRB (Proteintech #55113- 1- AP), NPRC (Novus Biologicals #NBP1- 31365), NanoLuc® Luciferase (R&D Systems #MAB10026), β- actin (Cell Signaling #4967).

Techniques: Expressing, Western Blot, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Discovery of another mechanism for the inhibition of particulate guanylyl cyclases by the natriuretic peptide clearance receptor.

doi: 10.1073/pnas.2307882120

Figure Lengend Snippet: Fig. 6. Coexpression of NPRC reduces ANP-evoked cGMP production in plasma membranes. Plasma membranes were made from HEK-293T cells transfected with either NPRA-flag plus pcDNA3-Clover control or NPRA-flag plus NPRC-flag. Plasma membranes were made without or with (SI Appendix, Fig. S3) phosphatase inhibitors. Co-expression of hNPRC along with hNPRA reduced the cGMP generated in the presence of 100 nM ANP (P = 0.0163, test for difference in slopes between NPRA and NPRA+NPRC; Šídák’s multiple comparisons test comparing NPRA vs. NPRA+NPRC are indicated on the graphs, ** P < 0.01.). Untransfected cells did not appear to generate cGMP. Western blot of membranes used in the ANP-stimulated cGMP assay.

Article Snippet: For western blotting the following primary antibodies were used: Flag (Sigma #F1804), NPRA (Novus Biologicals #NBP1- 31333), NPRB (Proteintech #55113- 1- AP), NPRC (Novus Biologicals #NBP1- 31365), NanoLuc® Luciferase (R&D Systems #MAB10026), β- actin (Cell Signaling #4967).

Techniques: Clinical Proteomics, Transfection, Control, Expressing, Generated, Western Blot

Journal: Frontiers in pharmacology

Article Title: Ginsenoside Rb1 Lessens Gastric Precancerous Lesions by Interfering With β-Catenin/TCF4 Interaction.

doi: 10.3389/fphar.2021.682713

Figure Lengend Snippet: FIGURE 6 | GRb1 inhibits hyper-proliferation of epithelial cells and accelerates apoptosis in GPL model rats. Representative images showing (A) PCNA expression and (C) Ki-67 expression as well as (E) TUNEL apoptotic cells in gastric tissue sections from each group (magnification ×100 and ×400). Semi-quantitative analysis of (B) PCNA protein expression levels and (D) Ki-67 protein expression levels as well as (F) TUNEL apoptosis ratio in each group (n 10). *p < 0.05 and **p < 0.01 vs. Normal group. #p < 0.05 and ##p < 0.01 vs. Model group. Data are presented as mean ± SEM. Abbreviations: GRb1, ginsenoside Rb1; GPL, gastric precancerous lesions; PCNA, proliferating cell nuclear antigen; TUNEL, TdT-mediated dUTP nick-end labeling; SEM, standard error of mean.

Article Snippet: Ki67 antibody was supplied by Novus Biologicals, LLC, United States (cat. no. NB500-170).

Techniques: Expressing, TUNEL Assay, End Labeling