Journal: European Journal of Human Genetics

Article Title: ADAMTS1 , MPDZ, MVD , and SEZ6: candidate genes for autosomal recessive nonsyndromic hearing impairment

doi: 10.1038/s41431-021-00913-x

Figure Lengend Snippet: Information on the variants found in the four candidate NSHI genes.

Article Snippet: The following primary antibodies were used: sheep anti-mouse ADAMTS1 (AF5867-SP; Novus Biologicals, Centennial, CO, USA), rabbit anti-mouse MPDZ (42-2700; Thermofisher Scientific, Waltham, MA, USA), rabbit anti-mouse MVD (PA5-22164; Thermofisher Scientific), and sheep anti-mouse SEZ6 (PA5-47683; Thermofisher Scientific).

Techniques:

Journal: European Journal of Human Genetics

Article Title: ADAMTS1 , MPDZ, MVD , and SEZ6: candidate genes for autosomal recessive nonsyndromic hearing impairment

doi: 10.1038/s41431-021-00913-x

Figure Lengend Snippet: Panel A : Structural model of the wild type ADAMTS1, MPDZ, MVD, and SEZ6 proteins. Panel B : Interacting bond of the wild type proteins for the four candidate genes. Panel C : Mutant type proteins highlighting the change due to the variant. The variants ADAMTS1 [p.(Ser135Ala)] and MPDZ [p.(Pro775Leu)] may result in a difference in interaction pattern with the solvent molecules. As the position of the variant residue is at the loop region, they do not involve a direct interaction, but the native fold of the protein may be affected. MVD [p.Pro379His] mutated protein shows a difference in interacting bond distance in the mutant type from the wild type and may perturb the amino acid side chain. The SEZ6 [p.Val698Ile] mutant protein displayed less interaction distance due to substitution of Val to Ile at position 698, while the SEZ6 wild type protein has a weak H-bond and hydrophobic interaction with Ile635 and Trp609. This variation could possibly change the orientation of the amino acid residue. Structures are displayed as ribbon while residue is represented by stick model. The structure illustrations were created with the PyMOL program. Black dotted lines represent hydrogen bonds.

Article Snippet: The following primary antibodies were used: sheep anti-mouse ADAMTS1 (AF5867-SP; Novus Biologicals, Centennial, CO, USA), rabbit anti-mouse MPDZ (42-2700; Thermofisher Scientific, Waltham, MA, USA), rabbit anti-mouse MVD (PA5-22164; Thermofisher Scientific), and sheep anti-mouse SEZ6 (PA5-47683; Thermofisher Scientific).

Techniques: Mutagenesis, Variant Assay, Solvent, Residue

Journal: European Journal of Human Genetics

Article Title: ADAMTS1 , MPDZ, MVD , and SEZ6: candidate genes for autosomal recessive nonsyndromic hearing impairment

doi: 10.1038/s41431-021-00913-x

Figure Lengend Snippet: A RNA expression of the four candidate genes in the cochlea and utricle during mouse development. RNA-sequencing data of hair cells (GFP+) and surrounding cells (GFP−) from the cochleae and utricles of mice expressing EGFP under the Pou4f3 promoter. Data were collected at four developmental stages: E16, P0, P4, and P7 and RNA expression is represented as normalized counts. Adamts1 , Mpdz , Mvd , and Sez6 were expressed in the hair cells and surrounding cells of the cochlea and utricle during the E16, P0, P4, and P7 stages. B RNA expression of the four candidate genes in the adult inner ear cells. The figure represents expression of the genes of interest in Deiters’ cells (Deiters), Pillar cells (Pillar), Inner Hair Cells (IHC), and Outer Hair Cells (OHC) of adult CBA/J mice. The four candidate genes Adamts1, Mpdz, Mvd , and Sez6 were expressed in the IHC, OHCs, and Deiter’s and Pillar cells. The y-axis represents the gene expression normalized to transcripts per million (TPM). The dataset was obtained from the GEO database (accession number GSE111347).

Article Snippet: The following primary antibodies were used: sheep anti-mouse ADAMTS1 (AF5867-SP; Novus Biologicals, Centennial, CO, USA), rabbit anti-mouse MPDZ (42-2700; Thermofisher Scientific, Waltham, MA, USA), rabbit anti-mouse MVD (PA5-22164; Thermofisher Scientific), and sheep anti-mouse SEZ6 (PA5-47683; Thermofisher Scientific).

Techniques: RNA Expression, RNA Sequencing, Expressing, Gene Expression

Journal: European Journal of Human Genetics

Article Title: ADAMTS1 , MPDZ, MVD , and SEZ6: candidate genes for autosomal recessive nonsyndromic hearing impairment

doi: 10.1038/s41431-021-00913-x

Figure Lengend Snippet: A Whole mount immunostaining of Adamts1, Mpdz, and Sez6 in wildtype mice at P12. Immunoreactivity was visualized with a fluorescently labeled secondary antibody (red) and F-actin was stained with phalloidin 488 nm (green). Immunolabeling of Adamts1 and Sez6 is observed in stereocilia as well as cytoplasm of outer hair cells. Mpdz is observed in the cytoplasmic region of hair cells. B Immunostaining of the organ of Corti at the apical, medial and basal turns of the cochlea at P4. C Immunostaining of Mvd in wildtype mice at P1, P4, and P28. Immunoreactivity of Mvd was visualized with a fluorescently labeled secondary antibody (green), F-actin was stained with rhodamine-phalloidin (red) and nuclear bodies were stained with DAPI (blue). The anti-neurofilament (NF-200, purple) was used to mark the neurons. Immunolabeling of Mvd is observed in spiral ganglion cells (SG). HP Habenula perforata, OC Organ of Corti.

Article Snippet: The following primary antibodies were used: sheep anti-mouse ADAMTS1 (AF5867-SP; Novus Biologicals, Centennial, CO, USA), rabbit anti-mouse MPDZ (42-2700; Thermofisher Scientific, Waltham, MA, USA), rabbit anti-mouse MVD (PA5-22164; Thermofisher Scientific), and sheep anti-mouse SEZ6 (PA5-47683; Thermofisher Scientific).

Techniques: Immunostaining, Labeling, Staining, Immunolabeling

Journal: European Journal of Human Genetics

Article Title: ADAMTS1 , MPDZ, MVD , and SEZ6: candidate genes for autosomal recessive nonsyndromic hearing impairment

doi: 10.1038/s41431-021-00913-x

Figure Lengend Snippet: Functions, expression and animal model phenotypes for the four NSHI candidate genes.

Article Snippet: The following primary antibodies were used: sheep anti-mouse ADAMTS1 (AF5867-SP; Novus Biologicals, Centennial, CO, USA), rabbit anti-mouse MPDZ (42-2700; Thermofisher Scientific, Waltham, MA, USA), rabbit anti-mouse MVD (PA5-22164; Thermofisher Scientific), and sheep anti-mouse SEZ6 (PA5-47683; Thermofisher Scientific).

Techniques: Expressing, Animal Model, Protein-Protein interactions, Membrane

Journal: PLoS ONE

Article Title: Breast Cancer Cells Induce Stromal Fibroblasts to Secrete ADAMTS1 for Cancer Invasion through an Epigenetic Change

doi: 10.1371/journal.pone.0035128

Figure Lengend Snippet: Relative gene expression of serglycin and ADAMTS1.

Article Snippet: For the purpose of neutralizing ADAMTS1 activity, 0.3–7.5 μg of mouse monoclonal anti-human ADAMTS1 antibody (R&D Systems) or mouse IgG 2B (R&D Systems) was added to 100 μl of 100% confluent fibroblast-cultured medium.

Techniques: Gene Expression

Journal: PLoS ONE

Article Title: Breast Cancer Cells Induce Stromal Fibroblasts to Secrete ADAMTS1 for Cancer Invasion through an Epigenetic Change

doi: 10.1371/journal.pone.0035128

Figure Lengend Snippet: (A) ADAMTS1 mRNA levels in indicated NAF derivatives were analyzed and shown gradually increased in NAF 200N.E4 from P0 to P3 and maintained from P3 to P5. (B) MeDIP indicates that ADAMTS1 promoter-associated DNA methylation level was similar in NAF 200N.P10, CAF 199C.P10 and NAF 200N.E4.P3 cells. (C) Bisulfite sequencing indicates that ADAMTS1 promoter in CAF 199C.P10, NAF 200N.P10 and NAF 200N.E4.P3 cells was hypomethylated. Closed circle: methylated cytosine. (D) ADAMTS1 promoter-associated H3K27me3 and EZH2 binding was decreased in CAF 199C.P10 and NAF 200N.E4.P3 cells. (B and D) MeDIP (B) or ChIP assays (D) using antibody against H3ace, H3K4me3, H3K9me3, H3K27me3, H3K36me3, H3K79me3 or EZH2 was performed in NAF 200N.P10 (white bars), CAF 199C.P10 (black bars) and NAF 200N.E4.P3 (gray bars) cells, followed by real-time PCR analysis. (E) ADAMTS1 promoter-associated H3K27me3 and EZH2 binding were decreased in NAF 200N.E4.P3 and maintained through passages to NAF 200N.E4.P5. ChIP assays using antibody against H3K27me3 (black bars) or EZH2 (white bars) were performed in cells indicated, followed by real-time PCR analysis. (B, D and E) PCR amplification was performed with DNA primers against the ADAMTS1 promoter region from −431 bp to −217 bp. Data are shown as mean ± SD from triplicate experiments. Statistical significance was evaluated by Student's t-test. * P <0.05. NS: No significant difference.

Article Snippet: For the purpose of neutralizing ADAMTS1 activity, 0.3–7.5 μg of mouse monoclonal anti-human ADAMTS1 antibody (R&D Systems) or mouse IgG 2B (R&D Systems) was added to 100 μl of 100% confluent fibroblast-cultured medium.

Techniques: Methylated DNA Immunoprecipitation, DNA Methylation Assay, Methylation Sequencing, Methylation, Binding Assay, Real-time Polymerase Chain Reaction, Amplification

Journal: PLoS ONE

Article Title: Breast Cancer Cells Induce Stromal Fibroblasts to Secrete ADAMTS1 for Cancer Invasion through an Epigenetic Change

doi: 10.1371/journal.pone.0035128

Figure Lengend Snippet: (A) Sequestration of the ADAMTS1 activity in the conditional medium derived from CAF 199C.P10 using an anti-ADAMTS1 antibody reduced invasion ability of MDA-MB-468 cells (left) and MDA-MB-231 cells (right) in an anti-ADAMTS1 Ab dose-dependent manner. (B) Neutralization of the ADAMTS1 activity in the conditional medium derived from NAF 200N.E4.P3 using an anti-ADAMTS1 antibody also reduced invasion ability of MDA-MB-468 cells (left) and MDA-MB-231 cells (right). (C) Western shows the efficient inhibition of ADAMTS1 protein level by three independent shRNAs. (D) The conditional medium derived from CAF 199C.P10 depleted of ADAMTS1 by shRNAs exhibited significantly lower activity for invasion of MDA-MB-468 cells (left) and MDA.MB-231 cells (right). (E) The conditional media derived from NAF 200N.P10 with exogenous ADAMTS1 expression (left, western) enhanced invasion of MDA-MB-468 cells (middle) and MDA-MB-231 cells (right). Data are shown as mean ± SD from triplicate experiments. Statistical significance was evaluated by Student's t-test. * P <0.05.

Article Snippet: For the purpose of neutralizing ADAMTS1 activity, 0.3–7.5 μg of mouse monoclonal anti-human ADAMTS1 antibody (R&D Systems) or mouse IgG 2B (R&D Systems) was added to 100 μl of 100% confluent fibroblast-cultured medium.

Techniques: Activity Assay, Derivative Assay, Neutralization, Western Blot, Inhibition, Expressing

Journal: PLoS ONE

Article Title: Breast Cancer Cells Induce Stromal Fibroblasts to Secrete ADAMTS1 for Cancer Invasion through an Epigenetic Change

doi: 10.1371/journal.pone.0035128

Figure Lengend Snippet: (A) A scatter dot plot of ADAMTS1 mRNA levels in CAFs assessed using quantitative real-time RT-PCR analysis. The ADAMTS1 mRNA levels in CAFs of patients with lymph node metastasis (n = 27) were significantly higher than in those of patients without lymph node metastasis (n = 22) (P = 0.0003). Data are shown as mean ± SD of triplicate samples. Statistical significance was evaluated by Student's t-test. *** P<0.001. (B) The relationship between ADAMTS1 expression and lymph node metastasis was analyzed using Fisher's exact test. ADAMTS1 expression levels in CAFs derived from patients with ≥60% lymph node metastasis were significantly higher than those of patients with ≤60% lymph node metastasis or with zero lymph node metastasis ( P = 0.001). Relative quantity (RQ) of ADAMTS1 expression compared to beta-actin expression: +, 0.001≤RQ≤0.025; ++, 0.025

Article Snippet: For the purpose of neutralizing ADAMTS1 activity, 0.3–7.5 μg of mouse monoclonal anti-human ADAMTS1 antibody (R&D Systems) or mouse IgG 2B (R&D Systems) was added to 100 μl of 100% confluent fibroblast-cultured medium.

Techniques: Quantitative RT-PCR, Expressing, Derivative Assay

Journal: PLoS ONE

Article Title: Breast Cancer Cells Induce Stromal Fibroblasts to Secrete ADAMTS1 for Cancer Invasion through an Epigenetic Change

doi: 10.1371/journal.pone.0035128

Figure Lengend Snippet: (A–C) Quantitative real-time RT-PCR analysis revealed that ADAMTS1 mRNA levels were higher in CAFs than in the corresponding NAFs in ten breast cancer patients (A), and that ADAMTS1 mRNA levels in CAF 199C.P10 and NAF 200N.E4.P3 were higher than in NAF 200N.P10 and 200N.E1-E3.P3 (B). Similar results are shown using the pairs of CAF 244C/NAF 245N and CAF 259C/NAF 260N (C). (D) The ADAMTS1 protein level in NAF 200N.E4.P3, but not NAF 200N.E1-E3.P3, was enhanced to the similar level in CAF 199C.P10, compared to NAF 200N.P10. (E) ELISA indicated that the ADAMTS1 protein level in cultured medium derived from NAF 200N.E4.P3 was higher than NAF 200N.E1-E3.P3 and NAF 200N.P10. (F) ADAMTS1 mRNA level in SK-BR-3 cell-precocultured NAF 200N.S4.P3 was also higher than in NAF 200N.P10 and 200N.S1-S3.P3. Data are shown as mean ± SD from triplicate experiments. Statistical significance was evaluated by Student's t-test. * P <0.05.

Article Snippet: For the purpose of neutralizing ADAMTS1 activity, 0.3–7.5 μg of mouse monoclonal anti-human ADAMTS1 antibody (R&D Systems) or mouse IgG 2B (R&D Systems) was added to 100 μl of 100% confluent fibroblast-cultured medium.

Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Cell Culture, Derivative Assay