Journal: PLoS ONE

Article Title: Plasma Proteomic Profiling in Hereditary Breast Cancer Reveals a BRCA1-Specific Signature: Diagnostic and Functional Implications

doi: 10.1371/journal.pone.0129762

Figure Lengend Snippet: List of analyzed samples.

Article Snippet: Rabbit monoclonal antibody against BRCA1 (clone D-20, Santa Cruz) was used at a final concentration of 1μg/ml.

Techniques: Mutagenesis

Journal: PLoS ONE

Article Title: Plasma Proteomic Profiling in Hereditary Breast Cancer Reveals a BRCA1-Specific Signature: Diagnostic and Functional Implications

doi: 10.1371/journal.pone.0129762

Figure Lengend Snippet: Analysis was performed on cells extracts. 4–15% precast SDS PAGE (Biorad) was used. 80 μg of proteins was loaded in each lane. BRCA1 and gelsolin expression was assayed in MCF-7 cell line stimulated with EGF at 3, 6 and 12h (A), in HCC1937 cell line stimulated with EGF at 3, 6 and 12h (B) and in MCF-7- BRCA1 interfered cell line. In each cell panel, left panel is representative data of western blot analysis; right panel is showing densitometric analysis for BRCA1 and gelsolin protein levels. Analysis was performed using three independent experiments. Data are mean ± SEM (N = 3). *p< 0.05. In each cell panel ɣ-tubulin blot shows equal amount of protein loading.

Article Snippet: Rabbit monoclonal antibody against BRCA1 (clone D-20, Santa Cruz) was used at a final concentration of 1μg/ml.

Techniques: SDS Page, Expressing, Western Blot

Journal: PLoS ONE

Article Title: Plasma Proteomic Profiling in Hereditary Breast Cancer Reveals a BRCA1-Specific Signature: Diagnostic and Functional Implications

doi: 10.1371/journal.pone.0129762

Figure Lengend Snippet: MCF-7 (panel A) and HCC1937 (panel B) cells were lysed and subjected to immunoprecipitation with the appropriate antibody as described in “Materials and Methods.” The immunoprecipitates were separated by SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. Total cells extract was used as positive control (input). The immunoblot was probed with rabbit anti-ATF1 antisera at 1 mg/ml (C-20 Santa Cruz.) and developed by enhanced chemiluminescence.(C) BRCA1 levels in MCF-7 and HCC1937 human breast cancer cells lines.

Article Snippet: Rabbit monoclonal antibody against BRCA1 (clone D-20, Santa Cruz) was used at a final concentration of 1μg/ml.

Techniques: Immunoprecipitation, Polyacrylamide Gel Electrophoresis, Membrane, Positive Control, Western Blot

Journal: Scientific Reports

Article Title: TNFα-signal and cAMP-mediated signals oppositely regulate melanoma- associated ganglioside GD3 synthase gene in human melanocytes

doi: 10.1038/s41598-019-51333-3

Figure Lengend Snippet: Synthetic pathway of sialic acid - containing glycosphingolipids, gangliosides and gene expression of glycosyltransferases in melanocytes and melanoma cell lines. ( a ) Glycosyltransferase s genes for the synthesis of gangliosides are shown in italics. ( b ) Gene expression levels of glycosyltransferases in 2 melanocyte cells and 7 melanoma cell lines were analyzed. mRNA expressions of GD3 synthase, GM2/GD2 synthase, and GM1/GD1b synthase were analyzed by qRT-PCR in melanocytes (HEMn-LP (LP) and HEMn-MP (MP)) and melanoma lines (SK-MEL-28, SK-MEL-37, MeWo, SK-MEL-23, SK-MEL-31, SK-MEL-173, and SK-MEL-130). mRNA expression levels of these glycosyltransferases were normalized by those of human β-actin gene. Data represent means ± standard deviation (s.d.) (n = 4). Statistical analysis was performed by the two-tailed Student’s t-test (* P < 0.05; ** P < 0.01).

Article Snippet: Antibodies used for detection of glycosyltransferases were as follows, GD3 Synthase (B-11) (a mouse monoclonal antibody) (Santa Cruz Biotechnology, Inc., Dallas, TX, USA); GM2/GD2 Synthase (C-5) (a mouse monoclonal antibody) (Santa Cruz Biotechnology, Inc.); B3GALT4 (GM1/GD1b synthase) Polyclonal Antibody (Proteintech Group, Inc., Rosemont, IL, USA).

Techniques: Gene Expression, Quantitative RT-PCR, Expressing, Standard Deviation, Two Tailed Test

Journal: Scientific Reports

Article Title: TNFα-signal and cAMP-mediated signals oppositely regulate melanoma- associated ganglioside GD3 synthase gene in human melanocytes

doi: 10.1038/s41598-019-51333-3

Figure Lengend Snippet: GD3 synthase gene upregulated by TNFα treatment or removal of dcAMP and IBMX from culture medium in melanocytes. ( a ) Time-course of mRNA expression levels of GD3 synthase, GM2/GD2 synthase, and GM1/GD1b synthase in melanocytes (LP) after adding TNFα to F10-A medium. Melanocytes were treated with TNFα (10 ng/mL) for 0–24 h. Then, mRNA expression levels were analyzed by qRT-PCR. mRNA expression levels of these glycosyltransferases were normalized by those of human GAPDH gene. Data represent means ± s.d. (n = 4). ( b ) Time-course of mRNA expression levels of glycosyltransferases in melanocytes (LP) after removing dcAMP (1 mM) and IBMX (0.1 mM) from F10-A medium. Melanocytes were cultured without dcAMP and IBMX for 0–24 h. Then, mRNA expression levels were analyzed by qRT-PCR. mRNA expression levels of these glycosyltransferases were normalized by those of human GAPDH gene. Data represent means ± s.d. (n = 4). Statistical analysis was performed by the two-tailed Student’s t-test (* P < 0.05; ** P < 0.01). ns = not significant. ( c ) Immunofluorescence staining of GD3 of melanocytes (LP). Results on immunocytostaining with anti-GD3 antibody (R24) and Alexa Fluor 594-anti-mouse IgG were shown. Stained GD3 was observed using a confocal microscope ( upper ), and in bright field ( lower ). (i) Melanocytes were cultured in F10-A medium and GD3 expression was analyzed. (ii) GD3 expression was analyzed at 6, 12, and 24 h after the addition of TNFα (10 ng/mL) to F10-A medium. (iii) GD3 expression was analyzed at 6, 12, and 24 h after the removal of dcAMP and IBMX from F10-A medium (F10 medium).

Article Snippet: Antibodies used for detection of glycosyltransferases were as follows, GD3 Synthase (B-11) (a mouse monoclonal antibody) (Santa Cruz Biotechnology, Inc., Dallas, TX, USA); GM2/GD2 Synthase (C-5) (a mouse monoclonal antibody) (Santa Cruz Biotechnology, Inc.); B3GALT4 (GM1/GD1b synthase) Polyclonal Antibody (Proteintech Group, Inc., Rosemont, IL, USA).

Techniques: Expressing, Quantitative RT-PCR, Cell Culture, Two Tailed Test, Immunofluorescence, Staining, Microscopy

Journal: Scientific Reports

Article Title: TNFα-signal and cAMP-mediated signals oppositely regulate melanoma- associated ganglioside GD3 synthase gene in human melanocytes

doi: 10.1038/s41598-019-51333-3

Figure Lengend Snippet: cAMP signal suppresses expression of GD3 synthase gene in melanocytes. ( a ) Melanocytes (LP) were cultured according to protocols 1~3. 1. Melanocytes were cultured in F10-A medium. 2. F10-A medium was exchanged with F10-A medium without dcAMP and IBMX (F10 medium) on day 5. 3. F10-A medium was exchanged with F10 medium on day 5, then dcAMP (1 mM) was added on day 6 and cultured for 12 h. ( b ) Melanocytes were cultured as shown in (a), and mRNA expression levels of GD3 synthase, GM2/GD2 synthase, and GM1/GD1b synthase were analyzed by qRT-PCR. mRNA expression levels of these glycosyltransferases were normalized by those of human GAPDH gene. Data represent means ± s.d. (n = 4). ( c ) Melanocytes (LP) were cultured according to protocols 1~4. 1. Melanocytes were cultured in F10-A medium. 2. F10-A medium was exchanged with F10 medium on day 5. 3. F10-A medium was exchanged with F10 medium on day 5, and dcAMP (1 mM) was added on day 6. 4. F10-A medium was exchanged with F10 medium on day 5. On day 6, α-MSH (100 nM) was added for 30 min, then medium was exchanged with F10 medium again. ( d ) Inhibitory effects of α-MSH on expression of the GD3 synthase gene in melanocytes. Melanocytes were cultured as shown in ( c ), and mRNA expression levels of GD3 synthase, GM2/GD2 synthase, and GM1/GD1b synthase were analyzed by qRT-PCR. mRNA expression levels of these glycosyltransferases were normalized by those of human GAPDH gene. Data represent means ± s.d. (n = 4). ( e ) Enhancing effects of a PKA inhibitor, H89, on expression of the GD3 synthase gene in melanocytes (LP). Melanocytes were cultured in F10-A medium for 4 days, and then an inhibitor of PKA, H89, was added. After treatment of melanocytes with H89 (10 μM) for 0, 8, 12, or 24 h, mRNA expression levels of GD3 synthase, GM2/GD2 synthase, and GM1/GD1b synthase were analyzed by qRT-PCR. mRNA expression levels of these glycosyltransferases were normalized by those of human GAPDH gene. Data represent means ± s.d. (n = 4). Statistical analysis was performed by two-tailed Student’s t-test (* P < 0.05; ** P < 0.01). ns = not significant.

Article Snippet: Antibodies used for detection of glycosyltransferases were as follows, GD3 Synthase (B-11) (a mouse monoclonal antibody) (Santa Cruz Biotechnology, Inc., Dallas, TX, USA); GM2/GD2 Synthase (C-5) (a mouse monoclonal antibody) (Santa Cruz Biotechnology, Inc.); B3GALT4 (GM1/GD1b synthase) Polyclonal Antibody (Proteintech Group, Inc., Rosemont, IL, USA).

Techniques: Expressing, Cell Culture, Quantitative RT-PCR, Two Tailed Test

Journal: Scientific Reports

Article Title: TNFα-signal and cAMP-mediated signals oppositely regulate melanoma- associated ganglioside GD3 synthase gene in human melanocytes

doi: 10.1038/s41598-019-51333-3

Figure Lengend Snippet: Combined treatment of melanocytes with TNFα and H89 or TNFα and removal of dcAMP further enhances expression of the GD3 synthase gene. ( a ) Melanocytes (LP) were cultured according to protocols 1~6. Melanocytes were cultured in F10-A medium for 3 days. Then, 1. melanocytes were cultured in F10-A medium for 24 h, 2. cultured in F10-A medium without dcAMP and IBMX (F10 medium) for 24 h, 3. treated with TNFα (10 ng/mL) for 24 h, 4. treated with H89 (10 μM) for 24 h, 5. Melanocytes were cultured in F10 medium with TNFα (10 ng/mL) for 24 h, and 6. cultured with TNFα (10 ng/mL) and H89 (10 μM) for 24 h. ( b ) Melanocytes were cultured according to protocols 1~6 as in ( a ). mRNA expression levels of GD3 synthase, GM2/GD2 synthase, and GM1/GD1b synthase were analyzed by qRT-PCR. mRNA expression levels of these glycosyltransferases were normalized by those of human GAPDH gene. Data represent means ± s.d. (n = 4). Statistical analysis was performed by the two-tailed Student’s t-test (** P < 0.01).

Article Snippet: Antibodies used for detection of glycosyltransferases were as follows, GD3 Synthase (B-11) (a mouse monoclonal antibody) (Santa Cruz Biotechnology, Inc., Dallas, TX, USA); GM2/GD2 Synthase (C-5) (a mouse monoclonal antibody) (Santa Cruz Biotechnology, Inc.); B3GALT4 (GM1/GD1b synthase) Polyclonal Antibody (Proteintech Group, Inc., Rosemont, IL, USA).

Techniques: Expressing, Cell Culture, Quantitative RT-PCR, Two Tailed Test

Journal: Scientific Reports

Article Title: TNFα-signal and cAMP-mediated signals oppositely regulate melanoma- associated ganglioside GD3 synthase gene in human melanocytes

doi: 10.1038/s41598-019-51333-3

Figure Lengend Snippet: Inhibition of IKK results in suppression of GD3 synthase gene expression induced by TNFα in melanocytes. ( a ) Melanocytes (LP) were cultured according to protocols 1~4. Melanocytes were cultured in F10-A medium for 4 days. Then, 1. melanocytes were cultured in F10-A medium for 24 h, 2. cultured with TNFα (10 ng/mL) for 24 h, 3. cultured with TNFα (10 ng/mL) and an IKK inhibitor, WDL (12.5 μM or 25 μM), for 24 h, and 4. cultured with WDL (25 μM). ( b ) After treatment of melanocytes with or without TNFα and WDL as in ( a ), mRNA expression levels of GD3 synthase, GM2/GD2 synthase, and GM1/GD1b synthase were analyzed by qRT-PCR. mRNA expression levels of these glycosyltransferases were normalized by those of human GAPDH gene. Data represent means ± s.d. (n = 4). Statistical analysis was performed by the two-tailed Student’s t-test (* P < 0.05; ** P < 0.01). ns = not significant.

Article Snippet: Antibodies used for detection of glycosyltransferases were as follows, GD3 Synthase (B-11) (a mouse monoclonal antibody) (Santa Cruz Biotechnology, Inc., Dallas, TX, USA); GM2/GD2 Synthase (C-5) (a mouse monoclonal antibody) (Santa Cruz Biotechnology, Inc.); B3GALT4 (GM1/GD1b synthase) Polyclonal Antibody (Proteintech Group, Inc., Rosemont, IL, USA).

Techniques: Inhibition, Gene Expression, Cell Culture, Expressing, Quantitative RT-PCR, Two Tailed Test

Journal: Scientific Reports

Article Title: TNFα-signal and cAMP-mediated signals oppositely regulate melanoma- associated ganglioside GD3 synthase gene in human melanocytes

doi: 10.1038/s41598-019-51333-3

Figure Lengend Snippet: dcAMP, TNFα, and H89 do not affect expression levels of glycosyltransferases, while WDL suppresses GD3 synthase gene expression in melanomas. Melanoma cell lines, SK-MEL-28, SK-MEL-37, and MeWo were treated with or without TNFα (10 ng/mL) ( a ), dcAMP (1 mM) ( b ), and H89 (10 μM) ( c ) for 24 h. Then, mRNA expression levels of GD3 synthase, GM2/GD2 synthase, and GM1/GD1b synthase were analyzed by qRT-PCR. mRNA expression levels of these glycosyltransferases were normalized by those of human GAPDH gene. Data represent means ± s.d. (n = 4). ( d ) Treatment of melanoma cell lines (SK-MEL-28, SK-MEL-23, MeWo, and SK-MEL-130) with WDL reduced expression levels of the GD3 synthase gene. After treatment with or without WDL (20 μM) for 24 h, mRNA expression s levels of GD3 synthase, GM2/GD2 synthase, and GM1/GD1b synthase were analyzed by qRT-PCR. mRNA expression levels of these glycosyltransferases were normalized by those of human GAPDH gene. Data represent means ± s.d. (n = 3–4). Statistical analysis was performed by the two-tailed Student’s t-test (* P < 0.05; ** P < 0.01). ns = not significant.

Article Snippet: Antibodies used for detection of glycosyltransferases were as follows, GD3 Synthase (B-11) (a mouse monoclonal antibody) (Santa Cruz Biotechnology, Inc., Dallas, TX, USA); GM2/GD2 Synthase (C-5) (a mouse monoclonal antibody) (Santa Cruz Biotechnology, Inc.); B3GALT4 (GM1/GD1b synthase) Polyclonal Antibody (Proteintech Group, Inc., Rosemont, IL, USA).

Techniques: Expressing, Gene Expression, Quantitative RT-PCR, Two Tailed Test

Journal: Scientific Reports

Article Title: TNFα-signal and cAMP-mediated signals oppositely regulate melanoma- associated ganglioside GD3 synthase gene in human melanocytes

doi: 10.1038/s41598-019-51333-3

Figure Lengend Snippet: TNFα and cAMP-PKA signal pathways exert opposite effects to regulate GD3 synthase gene expression in human melanocytes. The TNFα signal pathway upregulated expression of the GD3 synthase gene, while cAMP-PKA signaling suppressed its expression.

Article Snippet: Antibodies used for detection of glycosyltransferases were as follows, GD3 Synthase (B-11) (a mouse monoclonal antibody) (Santa Cruz Biotechnology, Inc., Dallas, TX, USA); GM2/GD2 Synthase (C-5) (a mouse monoclonal antibody) (Santa Cruz Biotechnology, Inc.); B3GALT4 (GM1/GD1b synthase) Polyclonal Antibody (Proteintech Group, Inc., Rosemont, IL, USA).

Techniques: Gene Expression, Expressing