mouse anti human muc2 antibody ccp58  (Novus Biologicals)

Journal: Scientific Reports

Article Title: Mucin 2 silencing promotes colon cancer metastasis through interleukin-6 signaling

doi: 10.1038/s41598-017-04952-7

Figure Lengend Snippet: Kaplan–Meier survival curves relative to mucin 2 ( MUC2) and interleukin 6 ( IL-6) gene expression in patients with colon cancer. ( a ) Disease-free survival (DFS) is analyzed with respect to MUC2 expression levels. ( b ) Disease-specific survival (DSS) and overall survival (OS) are analyzed with respect to IL-6 expression levels. The total number of patients in the low and high expression groups and P- values are shown. The high and low MUC2 expression groups were determined using the best cutoff value according to the PrognoScan database description. The x-axis represents time, and the y-axis represents DFS ( a ), DSS ( b ), and OS ( b ). ( c ) Immunohistochemical staining to determine the distribution of MUC2-positive cells (red), CD68-positive cells (red) and IL-6-positive cells (red) in paraffin-embedded specimens from AJCC stage IIA colon cancer patients (magnification, ×200). AJCC, American Joint Committee on Cancer. A stage IIA colon cancer specimen with high MUC2 expression and low IL-6 and CD68 expression (magnification, ×200) (upper). A stage IIA colon cancer specimen with low MUC2 expression and high IL-6 and CD68 expression (magnification, ×200) (lower). ( d ) Expression of MUC2 and differentiation type in stage IIA colon cancer as assessed by immunohistochemical (IHC) staining and scaling according to the immunoreactive score (IRS) of Remmele and Stegner, as follows: 0–1, negative expression; 2–3, weak expression; 4–8, mild expression; 9–12, strong expression. ( e ) Correlation between MUC2 and IL-6 expression in colon cancer cells of patients with stage IIA colon cancer (upper). Patients with a lower level of MUC2 expression specifically had higher immune cell infiltration (lower).

Article Snippet: After the sections were subjected to antigen retrieval in an autoclave, immunohistochemical staining was performed by incubating the sections overnight with a mouse anti-human MUC2 antibody (Ccp58; 1:100 dilution; Novocastra, Newcastle upon Tyne, England), a mouse anti-human IL-6 antibody (3G9; 1:100 dilution; Origene Technologies, Inc., Rockville, MD) and a mouse anti-human IL-68 antibody (clone KP-1; 1:100 dilution; code No. M0814; DAKO).

Techniques: Gene Expression, Expressing, Immunohistochemical staining, Staining, Immunohistochemistry

Journal: Scientific Reports

Article Title: Mucin 2 silencing promotes colon cancer metastasis through interleukin-6 signaling

doi: 10.1038/s41598-017-04952-7

Figure Lengend Snippet: MUC2 silencing in HT-29 and LS174T cells did not influence cell proliferation. ( a and b ) MUC2 protein expression was determined in human HT-29 and LS174T cells and MUC2 shRNA stable transfectants. The results of the western blot analysis of protein expression were obtained from three independent experiments. The bars represent the mean ± the SD. P, parental cells; shLuc, luciferase control; shMUC2-1.1, shMUC2-1.2, shMUC2-2.1 and shMUC2-2.2, MUC2-specific shRNAs 1 and 2, respectively. ( c and d ) The proliferation of HT-29 and LS174T cells and MUC2 shRNA stable transfectants was determined at 24, 48, and 72 h. NS, not significant; *P < 0.01; **P < 0.001; ***P < 0.0001.

Article Snippet: After the sections were subjected to antigen retrieval in an autoclave, immunohistochemical staining was performed by incubating the sections overnight with a mouse anti-human MUC2 antibody (Ccp58; 1:100 dilution; Novocastra, Newcastle upon Tyne, England), a mouse anti-human IL-6 antibody (3G9; 1:100 dilution; Origene Technologies, Inc., Rockville, MD) and a mouse anti-human IL-68 antibody (clone KP-1; 1:100 dilution; code No. M0814; DAKO).

Techniques: Expressing, shRNA, Western Blot, Luciferase, Control

Journal: Scientific Reports

Article Title: Mucin 2 silencing promotes colon cancer metastasis through interleukin-6 signaling

doi: 10.1038/s41598-017-04952-7

Figure Lengend Snippet: MUC2 silencing increased cell migration/metastasis and STAT3 silencing inhibited cell migration in HT-29 cell clones. ( a ) MUC2-silenced and STAT3-silenced HT-29 cells were examined using the wound-healing assay. ( b ) MUC2-silenced LS174T cells were examined using the wound-healing assay. The data represent quantitative results of the in vitro wound-healing assay at 48 h and are presented as the mean ± the SD of three independent experiments. ( c – e ) Hematoxylin and eosin staining of liver sections (magnification, ×40); macrometastases are indicated by arrows. Liver metastasis was observed in mice injected with colon cancer cells transfected with shLuc ( c ), shMUC2-1.2 ( d ) and shMUC2-2.2 ( e ). The boxed areas in ( c ), ( d ) and ( e ) are shown at higher magnification (×100). ( f ) The number of tumor nodules per field of liver in NOD/SCID mice was determined on postinjection day 14. The data are expressed as the mean ± the SD of two independent experiments. P, parental cells; shLuc, luciferase control; shMUC2-1.2 and shMUC2-2.2, MUC2-specific shRNAs 1 and 2, respectively; NS, not significant; *P < 0.01; **P < 0.001; ***P < 0.0001.

Article Snippet: After the sections were subjected to antigen retrieval in an autoclave, immunohistochemical staining was performed by incubating the sections overnight with a mouse anti-human MUC2 antibody (Ccp58; 1:100 dilution; Novocastra, Newcastle upon Tyne, England), a mouse anti-human IL-6 antibody (3G9; 1:100 dilution; Origene Technologies, Inc., Rockville, MD) and a mouse anti-human IL-68 antibody (clone KP-1; 1:100 dilution; code No. M0814; DAKO).

Techniques: Migration, Clone Assay, Wound Healing Assay, In Vitro, Staining, Injection, Transfection, Luciferase, Control

Journal: Scientific Reports

Article Title: Mucin 2 silencing promotes colon cancer metastasis through interleukin-6 signaling

doi: 10.1038/s41598-017-04952-7

Figure Lengend Snippet: Profiling phosphor-signaling networks in HT-29-derived cells after exogenous treatment with recombinant human IL-6. To detect intracellular signaling, the activation of important signaling components was observed using human Phospho-RTK Arrays. MUC2 suppression leads to decreased CREB phosphorylation ( a ) and increased STAT3 ( b ) and Chk2 phosphorylation ( a ) in HT-29 colon cancer cells exogenously supplemented with human rIL-6. ( c ) Bars represent the mean ± the SD. shMUC2-1.2, MUC2-specific shRNA 1.

Article Snippet: After the sections were subjected to antigen retrieval in an autoclave, immunohistochemical staining was performed by incubating the sections overnight with a mouse anti-human MUC2 antibody (Ccp58; 1:100 dilution; Novocastra, Newcastle upon Tyne, England), a mouse anti-human IL-6 antibody (3G9; 1:100 dilution; Origene Technologies, Inc., Rockville, MD) and a mouse anti-human IL-68 antibody (clone KP-1; 1:100 dilution; code No. M0814; DAKO).

Techniques: Derivative Assay, Recombinant, Activation Assay, Phospho-proteomics, shRNA

Journal: Scientific Reports

Article Title: Mucin 2 silencing promotes colon cancer metastasis through interleukin-6 signaling

doi: 10.1038/s41598-017-04952-7

Figure Lengend Snippet: Exogenous rIL-6 induces IL-6-dependent signaling in HT-29-derived cells. ( a ) Time-course and dose-response analysis of HT-29-derived cells with or without recombinant human IL-6 treatment. ( b – e ) Increased STAT3/Chk2 phosphorylation and decreased CREB phosphorylation were detected in MUC2-silenced HT-29 cells after exogenous treatment with recombinant human IL-6. ( b ) Western blot analysis of STAT3, Chk2, and CREB in HT-29-derived cells. MUC2-specific shRNA increased the phosphorylation levels of STAT3 ( c ) and Chk2 ( d ) and decreased the phosphorylation levels of CREB ( e ) in HT-29 cells treated with exogenous human rIL-6. The data represent the mean ± the SD of three independent experiments. STAT3 isoform expression appears as STAT3α (86 kDa) and STAT3β (79 kDa). P, parental cells; shLuc, luciferase control; shMUC2-1.2 and shMUC2-2.2, MUC2-specific shRNAs 1 and 2, respectively; NS, not significant; **P < 0.001; ***P < 0.0001.

Article Snippet: After the sections were subjected to antigen retrieval in an autoclave, immunohistochemical staining was performed by incubating the sections overnight with a mouse anti-human MUC2 antibody (Ccp58; 1:100 dilution; Novocastra, Newcastle upon Tyne, England), a mouse anti-human IL-6 antibody (3G9; 1:100 dilution; Origene Technologies, Inc., Rockville, MD) and a mouse anti-human IL-68 antibody (clone KP-1; 1:100 dilution; code No. M0814; DAKO).

Techniques: Derivative Assay, Recombinant, Phospho-proteomics, Western Blot, shRNA, Expressing, Luciferase, Control

Journal: Scientific Reports

Article Title: Mucin 2 silencing promotes colon cancer metastasis through interleukin-6 signaling

doi: 10.1038/s41598-017-04952-7

Figure Lengend Snippet: Expression of E-cadherin in HT-29-derived cells with or without recombinant human IL-6 treatment. HT-29-derived cells were treated with rIL-6 for 30 min. E-cadherin, vimentin, MUC2, and STAT3 expression levels were examined in IL-6-treated HT-29-derived cells by western blotting. ( a ) Loss of E-cadherin expression in MUC2-silenced HT-29 cells with or without recombinant human IL-6 treatment for 30 min. ( b ) Significantly reversed E-cadherin expression in STAT3-silenced HT-29 cells with or without recombinant human IL-6 treatment for 30 min. P, parental cells; shLuc, luciferase control; shMUC2-1.2 and shMUC2-2.2, MUC2-specific shRNAs 1 and 2, respectively; shSTAT3-1 and shSTAT3-2, STAT3-specific shRNAs 1 and 2, respectively; NS, not significant; **P < 0.001; ***P < 0.0001. ( c ) Proposed model depicting the effects of MUC2 and IL-6 treatment on cell migration and metastasis in HT-29 colon cancer cells. ( d ) Protein interaction network of MUC2, IL-6, STAT3, CREB1, CHEK2, CDH1, and TP53. The colored lines between the proteins indicate the various types of evidence demonstrating the interaction. The evidence for these interactions is derived from both experimental evidence (purple lines) and text-mining evidence (green lines). MUC2: mucin 2; TP53: Tumor protein p53; CREB1: cAMP responsive element-binding protein 1; CDH1: epithelial cadherin (E-cadherin); STAT3: Signal transducer and activator of transcription 3; IL-6: Interleukin 6; CHEK2: Checkpoint kinase 2.

Article Snippet: After the sections were subjected to antigen retrieval in an autoclave, immunohistochemical staining was performed by incubating the sections overnight with a mouse anti-human MUC2 antibody (Ccp58; 1:100 dilution; Novocastra, Newcastle upon Tyne, England), a mouse anti-human IL-6 antibody (3G9; 1:100 dilution; Origene Technologies, Inc., Rockville, MD) and a mouse anti-human IL-68 antibody (clone KP-1; 1:100 dilution; code No. M0814; DAKO).

Techniques: Expressing, Derivative Assay, Recombinant, Western Blot, Luciferase, Control, Migration, Binding Assay