Journal: Molecular Cancer

Article Title: Syndecan-1 is a novel molecular marker for triple negative inflammatory breast cancer and modulates the cancer stem cell phenotype via the IL-6/STAT3, Notch and EGFR signaling pathways

doi: 10.1186/s12943-017-0621-z

Figure Lengend Snippet: Expression of Syndecan-1 and the CSC marker CD44 in carcinoma tissues of IBC vs non-IBC patients, SUM-149 and SKBR3 cells. a Higher expression of Syndecan-1 mRNA level in carcinoma tissues of IBC ( n = 13) vs non-IBC ( n = 14). RQ values of mRNA expression are log2-transformed and normalized to values of normal tissues collected during reduction mammoplasty. Bars represent median with interquartile range. ** P < 0.01 as determined by Mann-Whitney U-test. b Representative fields of immunostaining ( brown color ) of Syndecan-1 and CD44 in paraffin embedded carcinoma tissue sections of triple negative IBC ( n = 13) and non-IBC ( n = 17) patients. A high density of cancer cells positive for CD44 and Syndecan-1 is observed in IBC vs non-IBC. c Pearson’s correlation between Syndecan-1 and CD44 expression in carcinoma tissues of IBC vs non-IBC. d A representative flow cytometric analysis for the expression of CD44 and Syndecan-1 in SUM-149 and SKBR3 cells. e Quantitative analysis of four subpopulations; CD44 (-) Syndecan-1 (-) , CD44 (+) Syndecan-1 (-) , CD44 (-) Syndecan-1 (+) and CD44 (+) Syndecan-1 (+) . Syndecan-1 is higher expressed in the CD44 (+) -enriched subset in SUM-149 cells than that in SKBR3 cells. Data represent mean ± SEM, n ≥ 3. ** P < 0.01, # P < 0.001 as determined by Student’s t-test. Data shown are a single experiment representative of three independent experiments

Article Snippet: The human IBC cell line SUM-149 (a kind gift from Dr. Bonnie Sloane, Wayne State University, Detroit, MI, USA) and the non-IBC cell line SKBR3 (ATCC/LGC Promochem, Wesel, Germany) were maintained in HAM’s-F12 and DMEM containing 10% FCS, 1% glutamine and 1% penicillin/streptomycin in a humidified atmosphere of 5% CO 2 at 37 °C, respectively.

Techniques: Expressing, Marker, Transformation Assay, MANN-WHITNEY, Immunostaining

Journal: Molecular Cancer

Article Title: Syndecan-1 is a novel molecular marker for triple negative inflammatory breast cancer and modulates the cancer stem cell phenotype via the IL-6/STAT3, Notch and EGFR signaling pathways

doi: 10.1186/s12943-017-0621-z

Figure Lengend Snippet: Syndecan-1 silencing suppresses CSC-related gene expression in SUM-149 and SKBR3 cells. Post Syndecan-1 knockdown, total RNA isolated from SUM-149 ( a ) and SKBR3 cells ( b ) was reverse transcribed into cDNA and subjected into qPCR. Data represent mean ± SEM, n ≥ 3. * P < 0.05, # P < 0.01 as determined by Student’s t-test

Article Snippet: The human IBC cell line SUM-149 (a kind gift from Dr. Bonnie Sloane, Wayne State University, Detroit, MI, USA) and the non-IBC cell line SKBR3 (ATCC/LGC Promochem, Wesel, Germany) were maintained in HAM’s-F12 and DMEM containing 10% FCS, 1% glutamine and 1% penicillin/streptomycin in a humidified atmosphere of 5% CO 2 at 37 °C, respectively.

Techniques: Gene Expression, Knockdown, Isolation, Reverse Transcription

Journal: Molecular Cancer

Article Title: Syndecan-1 is a novel molecular marker for triple negative inflammatory breast cancer and modulates the cancer stem cell phenotype via the IL-6/STAT3, Notch and EGFR signaling pathways

doi: 10.1186/s12943-017-0621-z

Figure Lengend Snippet: Syndecan-1 silencing attenuates the activation of STAT-3 and NFκB signaling pathways and downregulates protein expression of gp130 in SUM-149 and SKBR3 cells. Seventy-two hours post transfection total cell lysates of control and Syndecan-1 knockdown cells were collected, electrophoresed and immunoblotted. The membrane was probed with the indicated antibodies. a Western blot showing expression of p-STAT-3, STAT-3, p-NFκB, NFκB and gp130 upon Syndecan-1 silencing in SUM-149 and SKBR-3 cells. b Immunoblot band intensities were normalized to the total form of STAT-3, NFκB or tubulin expression. Data shown are a single experiment representative of three independent experiments. * P < 0.05, ** P < 0.01 and # P < 0.001 as determined by Student’s t-test

Article Snippet: The human IBC cell line SUM-149 (a kind gift from Dr. Bonnie Sloane, Wayne State University, Detroit, MI, USA) and the non-IBC cell line SKBR3 (ATCC/LGC Promochem, Wesel, Germany) were maintained in HAM’s-F12 and DMEM containing 10% FCS, 1% glutamine and 1% penicillin/streptomycin in a humidified atmosphere of 5% CO 2 at 37 °C, respectively.

Techniques: Activation Assay, Protein-Protein interactions, Expressing, Transfection, Control, Knockdown, Membrane, Western Blot

Journal: Molecular Cancer

Article Title: Syndecan-1 is a novel molecular marker for triple negative inflammatory breast cancer and modulates the cancer stem cell phenotype via the IL-6/STAT3, Notch and EGFR signaling pathways

doi: 10.1186/s12943-017-0621-z

Figure Lengend Snippet: Syndecan-1 is a modulator of EGFR expression and activation via Notch signaling in IBC. a Scatter plot shows a significant upregulation of EGFR mRNA in tissues of IBC ( n = 13) vs non-IBC ( n = 14). RQ values of EGFR mRNA expression in tissues of IBC vs non-IBC are log2-transformed and normalized to values of normal tissues collected during reduction mammoplasty. Bars represent median with interquartile range. * P < 0.05 as determined by Mann-Whitney U-test. b Pearson’s correlation between Syndecan-1 and EGFR mRNA expression in tissues of non-IBC tissues ( left panel ) and IBC ( right panel ). c EGFR mRNA and protein level expression in control and Syndecan-1-silenced SUM-149 and SKBR3 cells. ** P < 0.01 and # P < 0.001 as determined by Student’s t-test. d Expression of EGFR mRNA levels in control and Syndecan-1-silenced SUM-149 cells post GSI treatment. * P < 0.05 and ** P < 0.01 as determined by one-way ANOVA followed by Tukey’s multiple comparison test. e Colony formation post 10 ng/ml EGF treatment in control and Syndecan-1-silenced SUM-149 cells. ** P < 0.01 and # P < 0.001 as determined by one-way ANOVA followed by Tukey’s multiple comparison test. f Western blot analysis of the downstream signaling p-Akt (Ser473) of EGFR signaling in response to EGF stimulation in control and Syndecan-1-silenced SUM-149 cells. Data represent mean ± SEM, n ≥ 3. Data shown are a single experiment representative of three independent experiments

Article Snippet: The human IBC cell line SUM-149 (a kind gift from Dr. Bonnie Sloane, Wayne State University, Detroit, MI, USA) and the non-IBC cell line SKBR3 (ATCC/LGC Promochem, Wesel, Germany) were maintained in HAM’s-F12 and DMEM containing 10% FCS, 1% glutamine and 1% penicillin/streptomycin in a humidified atmosphere of 5% CO 2 at 37 °C, respectively.

Techniques: Expressing, Activation Assay, Transformation Assay, MANN-WHITNEY, Control, Comparison, Western Blot

Journal: The Journal of general virology

Article Title: Bovine viral diarrhea virus non-structural protein 5A interacts with NIK- and IKKbeta-binding protein.

doi: 10.1099/vir.0.020990-0

Figure Lengend Snippet: Fig. 1. Direct association of NIBP with NS5A by GST pull-down assay. LB9.K cells were transiently transfected with FLAG–NIBP. Either GST alone or GST–NS5A purified from E. coli was incubated with cell lysate containing NIBP. Bound proteins were precipitated by glutathione beads as described in Methods and detected by immunoblotting. Both NIBP and NS5A were verified by anti-FLAG (top panel) and anti-NS5A (middle panel) mAbs, showing that GST–NS5A, but not GST alone, associated with FLAG–NIBP. As a control, cell lysates were immunoblotted using anti-GST rabbit polyclonal antibody (bottom panel). IB, Immunoblot. Arrows indicate GST–NS5A and GST alone at approximately 82 and 26 kDa, respectively.

Article Snippet: Rabbit anti-human NIBP polyclonal antibody was from Imgenex.

Techniques: Pull Down Assay, Transfection, Purification, Incubation, Western Blot, Control

Journal: The Journal of general virology

Article Title: Bovine viral diarrhea virus non-structural protein 5A interacts with NIK- and IKKbeta-binding protein.

doi: 10.1099/vir.0.020990-0

Figure Lengend Snippet: Fig. 2. NIBP interacts with BVDV NS5A in mammalian cells. (a) c-Myc–NS5A of strain Nose from BVDV and FLAG-tagged NIBP were expressed in LB9.K cells and immunoprecipitated (IP) with anti-c-Myc or anti-FLAG antibody. Immunoprecipitates were subjected to immunoblotting (IB) to detect coprecipitated counterparts. As a negative control, an empty plasmid was used instead of the plasmid encoding FLAG–NIBP or c-Myc–NS5A. Anti-FLAG or anti-c-Myc did not recognize c-Myc-tagged NS5A and FLAG-tagged NIBP, respectively. (b) Endogenous NIBP in LB9.K cells infected with cp and ncp BVDV was immunoprecipitated with normal control rabbit IgG1 (lane 1) or anti-NIBP rabbit IgG (lane 2), and immunoprecipitates were analysed by immunoblotting with specific antibodies.

Article Snippet: Rabbit anti-human NIBP polyclonal antibody was from Imgenex.

Techniques: Immunoprecipitation, Western Blot, Negative Control, Plasmid Preparation, Infection, Control

Journal: The Journal of general virology

Article Title: Bovine viral diarrhea virus non-structural protein 5A interacts with NIK- and IKKbeta-binding protein.

doi: 10.1099/vir.0.020990-0

Figure Lengend Snippet: Fig. 3. Determination of the NS5A-binding region in NIBP. (a) Structure and functional domains of NIBP. (b) Full-length or deletion mutants of NIBP used in the study and the results of binding to NS5A. N-terminally FLAG-tagged NIBP mutants encoding the regions indicated were designated 1–1138, 1–786, 624–1138, 191–596 or 528–864, respectively. A summary of immunoprecipitation results is given on the right. (c) Each mutant or full-length NIBP was coexpressed with c-Myc–NS5A in LB9.K cells, immunoprecipitated with an anti-c-Myc antibody and analysed by immunoblotting with an anti-FLAG antibody. As a negative control, an empty plasmid was used instead of the plasmid encoding c-Myc–NS5A. The anti-c-Myc antibody did not recognize FLAG-tagged NIBP or its mutants. The arrow indicates the full-length NIBP from aa 1 to 1138 on the immunoblot.

Article Snippet: Rabbit anti-human NIBP polyclonal antibody was from Imgenex.

Techniques: Binding Assay, Functional Assay, Immunoprecipitation, Mutagenesis, Western Blot, Negative Control, Plasmid Preparation

Journal: The Journal of general virology

Article Title: Bovine viral diarrhea virus non-structural protein 5A interacts with NIK- and IKKbeta-binding protein.

doi: 10.1099/vir.0.020990-0

Figure Lengend Snippet: Fig. 4. NS5A colocalizes with NIBP in the ER membrane. LB9.K cells were transiently transfected with FLAG–NIBP expression vector and, 6 h later, were infected with cp or ncp BVDV strains at an m.o.i. of 2 and further incubated for 18 h. The transfected cells were fixed and immunostained with anti-NIBP rabbit polyclonal antibody, anti-Grp 78/Bip or anti-NS5A mAbs. Colocalization was observed by superimposition of green and red images. (a) Anti- NIBP (red) and anti-NS5A (green); (b) anti-NIBP (green) and anti- Grp78/Bip (red).

Article Snippet: Rabbit anti-human NIBP polyclonal antibody was from Imgenex.

Techniques: Membrane, Transfection, Expressing, Plasmid Preparation, Infection, Incubation

Journal: The Journal of general virology

Article Title: Bovine viral diarrhea virus non-structural protein 5A interacts with NIK- and IKKbeta-binding protein.

doi: 10.1099/vir.0.020990-0

Figure Lengend Snippet: Fig. 5. NS5A inhibits TNF-a- and poly(I : C)- induced NF-kB activation. Effect of NS5A protein on TNF-a-induced NF-kB activation by luciferase reporter-gene assay. Either HEK293 (a) or LB9.K (b, c) cells were cotransfected with pNF-kB-Luc and pRL-TK reporter plasmids together with empty vectors (pCAGGS or pFLAG–CMV2) or pFLAG– CMV2–NIBP or/and pCAGGS–NS5A expres- sion plasmids, as indicated. At 24 h after transfection, the cells were treated with either TNF-a (10 ng ml”1) for 6 h or poly(I : C) (LB9.K cells only) for 12 h, and NF-kB activity was measured as described in Methods. The results shown are representative of three independent experiments done in duplicate, and values are means±SEM. *P,0.05 (sig- nificantly different from the control; Student’s t-test).

Article Snippet: Rabbit anti-human NIBP polyclonal antibody was from Imgenex.

Techniques: Activation Assay, Luciferase, Reporter Gene Assay, Transfection, Activity Assay, Control

Journal: The Journal of general virology

Article Title: Bovine viral diarrhea virus non-structural protein 5A interacts with NIK- and IKKbeta-binding protein.

doi: 10.1099/vir.0.020990-0

Figure Lengend Snippet: Fig. 6. Inhibition of NIBP by siRNA-enhanced viral replication. (a) LB9.K cells (106) were treated with either mock-transfected (–), NIBP-specific (+) or scrambled control non-specific (Scr) siRNAs at a final concentration of 66 nM. In the top panel, an immunoblot probed with anti-NIBP antibody is shown. For the bottom panel, the blot was probed with b-actin internal control. The experiment was repeated more than three times with similar results. (b) At 24 h post-transfection with siRNAs as described above, LB9.K cells were mock infected or infected with cp and ncp BVDV at an m.o.i. of 2. Cells were further incubated for 24 h and BVDV RNA level was determined by real-time PCR analysis. Values are expressed relative to the level of GAPDH RNA. (c) At the same time, the culture supernatants from the cp and ncp BVDV-infected LB9.K cells were collected and used to infect MDBK cells for 50 % tissue culture infective dose (TCID50) assay as described in Methods. The results shown are representative of three independent experiments done in duplicate, and values are means±SEM. *P,0.05 (significantly different from the control; Student’s t-test).

Article Snippet: Rabbit anti-human NIBP polyclonal antibody was from Imgenex.

Techniques: Inhibition, Transfection, Control, Concentration Assay, Western Blot, Infection, Incubation, Real-time Polymerase Chain Reaction, TCID50 Assay

Journal: The Journal of general virology

Article Title: Bovine viral diarrhea virus non-structural protein 5A interacts with NIK- and IKKbeta-binding protein.

doi: 10.1099/vir.0.020990-0

Figure Lengend Snippet: Fig. 7. Schematic layout of NIBP–NS5A interaction and the NF- kB pathway. Schematic layout showing a model of BVDV NS5A- mediated TNF-a-induced inhibition of the NF-kB signalling pathway. The inhibition appears to occur at the level of NIK and the IKK complex.

Article Snippet: Rabbit anti-human NIBP polyclonal antibody was from Imgenex.

Techniques: Inhibition