Journal: eLife

Article Title: Rab7-dependent regulation of goblet cell protein CLCA1 modulates gastrointestinal homeostasis

doi: 10.7554/elife.89776

Figure Lengend Snippet: Figure 2. Goblet cells of intestine display dominant expression of Rab7. (A, B) Confocal imaging of healthy and dextran sulfate sodium (DSS)-treated mice (7 d) colon sections showing Rab7 (red) expression within goblet cells (UEA1-FITC) marked with asterisk. Inset shows zoomed areas of the image (scale bar = 20 µm). Graph shows Rab7 fluorescence intensity within UEA1 positive goblet cells measured within region of interest (ROI). 20 goblet cells were selected randomly from three different fields of each mouse (n = 3). (C, D) Representative co-immunofluorescence images of human ulcerative colitis (UC) and control colon biopsy sections stained for Rab7 (red) and Muc2 (green) for goblet cells (marked with asterisk) (scale bar = 50 µm). Inset

Article Snippet: 8K206 Cell line (human) HEK293T Rab7-/(human) This paper This is a new reagent (details in ‘Material and methods’) Transfected construct (human) pSNMUC2- MG plasmid Prof. Gunnar Hansson Godl et al., 2002 Antibody Anti- Rab7a (rabbit polyclonal) Sigma- Aldrich Cat# R4779 WB: 1:5000 IHC: 1:400 (indirect) IF: 1:400 (indirect) IEM: 1:1000 Antibody Anti- Rab7a (mouse monoclonal) CST Cat# 95746 IP: 1:50 IF: 1:400 Antibody Anti- CLCA1 (rabbit monoclonal) Abcam Cat# ab180851 WB: 1:5000- 1:20,000 IHC: 1:100 IF: 1:100 Antibody Anti- Muc2 (mouse monoclonal) Santa Cruz Cat# sc- 515032 IF: 1:200 Antibody Anti- GAPDH (mouse monoclonal) Invitrogen Cat# 39- 8600; RRID:AB_2533438 WB: 1:2000 Antibody Anti-β-Actin (rabbit polyclonal) CST Cat# 4970S WB: 1:20,000 Antibody Anti- rabbit HRP (mouse monoclonal) Jackson ImmunoResearch Laboratories Cat# 211- 032- 171; RRID:AB_2339149 WB: 1:20,000 Antibody Anti- mouse HRP (goat polyclonal) Invitrogen Cat# 31430; RRID:AB_228307 WB: 1:5000 Antibody Biotin SP anti- rabbit antibody (goat polyclonal) Jackson ImmunoResearch Laboratories Cat# 111- 065- 003; RRID:AB_2337959 IF: 1:500 Antibody Anti- rabbit Alexa Flour 488 (goat polyclonal) Jackson ImmunoResearch Laboratories Cat# 111- 545- 003; RRID:AB_2338046 IF: 1:400 Antibody Anti- mouse Alexa Flour 488 (goat polyclonal) Jackson ImmunoResearch Laboratories Cat# 115- 545- 003; RRID:AB_2338840 IF: 1:400 Antibody Anti- rabbit Cy5 (goat polyclonal) Jackson ImmunoResearch Laboratories Cat# 111- 175- 144; RRID:AB_2338013 IF: 1:400 Antibody Anti- mouse Cy5 (goat polyclonal) Jackson ImmunoResearch Laboratories Cat# 111- 175- 146; RRID:AB_2338713 IF: 1:400 Antibody 18 nm Colloidal Gold- AffinityPure Anti- Rabbit (goat polyclonal) Jackson ImmunoResearch Laboratories Cat# 111- 215- 144; RRID:AB_2338017 IEM: 1:50 Recombinant DNA reagent pSpCas9(BB)- 2A- Puro addgene 62988 Sequence- based reagent ON- TARGETplus Smart Pool Mouse Rab7a siRNA Dharmacon L- 040859- 02- 0005 Sequence- based reagent ON- TARGETplus Smart Pool Human Rab7a siRNA Dharmacon 010388- 00- 0005 Sequence- based reagent ON- TARGETplus Non- targeting Control Pool Dharmacon D- 001810- 10- 20 Sequence- based reagent EUB338- Alexa Fluor 647 GCT GCC TCC CGT AGG AGT IDT Peptide, recombinant protein Streptavidin Cy2 Jackson ImmunoResearch Laboratories 16220084 Gaur et al. eLife 2023;13:RP89776.

Techniques: Expressing, Imaging, Fluorescence, Immunofluorescence, Control, Staining

Journal: Frontiers in Immunology

Article Title: MUC2 expression modulates immune infiltration in colorectal cancer

doi: 10.3389/fimmu.2024.1500374

Figure Lengend Snippet: Sequence of each gRNA designed for MUC2 K.O.

Article Snippet: After removing the blocking media, the spheroids were incubated for 45 min with primary antibody diluted in blocking media (mouse anti-human MUC2 antibody (CCP58) (Novus #NBP2-25221) diluted at 1:100 or rabbit anti-human Muc5B (Thermo Fisher Scientific, #PA5-82342) diluted at 1:25 or mouse anti-human Muc5AC (Thermo Fisher Scientific, #MA5-12178)) ( ).

Techniques: Sequencing

Journal: Frontiers in Immunology

Article Title: MUC2 expression modulates immune infiltration in colorectal cancer

doi: 10.3389/fimmu.2024.1500374

Figure Lengend Snippet: Primers used for PCR and sanger sequencing of MUC2 exon 2.

Article Snippet: After removing the blocking media, the spheroids were incubated for 45 min with primary antibody diluted in blocking media (mouse anti-human MUC2 antibody (CCP58) (Novus #NBP2-25221) diluted at 1:100 or rabbit anti-human Muc5B (Thermo Fisher Scientific, #PA5-82342) diluted at 1:25 or mouse anti-human Muc5AC (Thermo Fisher Scientific, #MA5-12178)) ( ).

Techniques: Sequencing

Journal: Frontiers in Immunology

Article Title: MUC2 expression modulates immune infiltration in colorectal cancer

doi: 10.3389/fimmu.2024.1500374

Figure Lengend Snippet: (A) Western blot of full-length MUC2 protein (540 kDa) and β-actin (housekeeping gene, 43 kDa) in three different cell lines LS-174T, HT-29, and LoVo grown in 2D ( N = 3 biological replicates). (B) Quantification of band intensity of MUC2 protein in those three cells lines grown in 2D. MUC2 is present in abundance in LS-174T, with significantly less expression in HT-29 ( p = 0.0019) (unpaired t -test), and no expression in LoVo cell line is observed. (C) Relative mRNA expression of MUC2 analyzed by Q-RT-PCR ( N = 3 biological replicates) in the three cell lines grown in 2D. MUC2 expression is strongest in LS-174T compared to HT-29 ( p = 0.0002) (unpaired t -test) and no expression in LoVo is observed. (D) Western blot of full length MUC2 protein (540kDa) and β-actin (43 kDa) in three different cell lines LS-174T, HT-29, and LoVo grown in 3D ( N = 3 biological replicates). (E) Quantification of band intensity of MUC2 protein in those three cell lines grown in 2D. MUC2 is present in abundance in LS-174T, with a significantly less expression in HT-29 ( p < 0.0001) (unpaired t -test), and no expression in LoVo cell line is observed. (F) Relative mRNA expression of MUC2 analyzed by Q-RT-PCR ( N = 3 biological replicates) in the three cell lines grown in 2D. MUC2 expression is strongest in LS-174T compared to HT-29 ( p = 0.0002) (unpaired t -test), and no expression in LoVo is observed. *p < 0.05, **p < 0.01, *** p < 0.001, and ****p < 0.0001.

Article Snippet: After removing the blocking media, the spheroids were incubated for 45 min with primary antibody diluted in blocking media (mouse anti-human MUC2 antibody (CCP58) (Novus #NBP2-25221) diluted at 1:100 or rabbit anti-human Muc5B (Thermo Fisher Scientific, #PA5-82342) diluted at 1:25 or mouse anti-human Muc5AC (Thermo Fisher Scientific, #MA5-12178)) ( ).

Techniques: Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Frontiers in Immunology

Article Title: MUC2 expression modulates immune infiltration in colorectal cancer

doi: 10.3389/fimmu.2024.1500374

Figure Lengend Snippet: (A) Western blot of full-length MUC2 protein (540 kDa) and β-actin (43 kDa) in different cell lines LS-174T CTRL, LS-174T MUC2 K.O., HT-29 CTRL, and HT-29 MUC2 K.O. grown in 2D ( N = 3 biological replicates). (B) Western blot of full-length MUC2 protein (540 kDa) and β-actin (43 kDa) in different cell lines LS-174T CTRL, LS-174T MUC2 K.O., HT-29 CTRL, and HT-29 MUC2 K.O. grown in 2D ( N = 3 biological replicates). (C) Representative images of spheroids of HT-29 and LS-174T CTRL and MUC2 K.O. immunostaining with mouse anti-hMUC2 antibody (green) and counterstained with DAPI. Images acquired on confocal LS780. Scale bar, 100 µm.

Article Snippet: After removing the blocking media, the spheroids were incubated for 45 min with primary antibody diluted in blocking media (mouse anti-human MUC2 antibody (CCP58) (Novus #NBP2-25221) diluted at 1:100 or rabbit anti-human Muc5B (Thermo Fisher Scientific, #PA5-82342) diluted at 1:25 or mouse anti-human Muc5AC (Thermo Fisher Scientific, #MA5-12178)) ( ).

Techniques: Western Blot, Immunostaining

Journal: Frontiers in Immunology

Article Title: MUC2 expression modulates immune infiltration in colorectal cancer

doi: 10.3389/fimmu.2024.1500374

Figure Lengend Snippet: (A) HT-29 CTRL and HT-29 MUC2 K.O. growth in 2D culture setup monitored by plate reader for 7 days by GFP intensity ( N = 3 biological replicates in 30 technical replicates each). HT-29 CTRL grew significantly faster than HT-29 MUC2 K.O. ( p = 0.0011) (unpaired t -test). (B) LS-174T CTRL and LS-174T MUC2 K.O. growth in 2D culture setup monitored by plate reader for 7 days by GFP intensity ( N = 3 biological replicates in 30 technical replicates each). No significant difference was observed (unpaired t -test). (C) HT-29 CTRL and HT-29 MUC2 K.O. growth in 2D culture setup monitored by plate reader for 7 days by GFP intensity ( N = 3 biological replicates in 30 technical replicates each). No significant difference was observed (unpaired t -test). (D) LS-174T CTRL and LS-174T MUC2 K.O. growth in 2D culture setup monitored by plate reader for 7 days by GFP intensity ( N = 3 biological replicates in 30 technical replicates each). LS-174T CTRL grew significantly faster than LS-174T MUC2 K.O. ( p = 0.0134) (unpaired t -test). (E) Representative images of day 5 spheroids acquired with a Zeiss CD7 fluorescent microscope of GFP and oblique of spheroids of HT-29 and LS-174T CTRL or MUC2 K.O. Scale of 200 µm. (F) Roundness analysis of HT-29 ( N = 13 each, biological replicates) and LS-174T ( N = 25 each, biological replicates) of CTRL and MUC2 K.O. performed by analysis with Zen blue software of area and perimeter of each spheroid. No significant difference is observed between HT-29 CTRL and HT-29 MUC2 K.O., but LS-174T MUC2 K.O. is significantly rounder than LS-174T CTRL (unpaired t -test). **p < 0.01; ns, not significant.

Article Snippet: After removing the blocking media, the spheroids were incubated for 45 min with primary antibody diluted in blocking media (mouse anti-human MUC2 antibody (CCP58) (Novus #NBP2-25221) diluted at 1:100 or rabbit anti-human Muc5B (Thermo Fisher Scientific, #PA5-82342) diluted at 1:25 or mouse anti-human Muc5AC (Thermo Fisher Scientific, #MA5-12178)) ( ).

Techniques: Microscopy, Software

Journal: Frontiers in Immunology

Article Title: MUC2 expression modulates immune infiltration in colorectal cancer

doi: 10.3389/fimmu.2024.1500374

Figure Lengend Snippet: (A) HT-29 and LS-174T, CTRL and MUC2 K.O. growth and death monitored by GFP expression over 7 days in 2D and 9 days in 3D culture set up. 0 or 500 E/A PBMCs were added on day 2 for 2D and 0 or 1,000 E/A PBMCs were added on day 5 for 3D co-culture setup. Data are presented as relative to the same cell line without co-culture with PBMCs (0 E/A PBMCs) ( N = 18, six technical replicates of three biological replicates). Significant differences between HT-29 and LS-174T cell lines were calculated at day 7 in 2D and 3D by Student’s t -test. (B) HT-29 CTRL and MUC2 K.O. spheroids area of IN fraction spheroids post-rinse after co-culture for 2 days with E/A PBCMs relative to the area of IN fraction after rinsing without co-culture. After co-culture with 2,000 or 4,000 PBMC, the area of IN fraction spheroids is significantly smaller in HT-29 MUC2 K.O. than in HT-29 CTRL ( N = 12, six technical replicates of two biological replicates) (unpaired t -test). (C) Representative images of IN fraction spheroids post-rinse after co-culture with 4,000 E/A PBMCs of HT-29 CTRL and HT-29 MUC2 K.O. Images were acquired for oblique and GFP expression on Zeiss CD7, and the area was analyzed with Imaris software. Scale bar, 200 µm. (D) LS-174T CTRL and MUC2 K.O. spheroid area of IN fraction spheroids post-rinse after co-culture for 2 days with E/A PBCMs relative to the area of IN fraction after rinsing without co-culture. No significant difference was observed between LS-174T CTRL and MUC2 K.O. at any number of E/A PBMC co-culture ( N = 12, two biological of six technical replicates) (unpaired t -test). (E) Representative images of IN fraction spheroids post-rinse after co-culture with 4,000 E/A PBMCs of LS-174T CTRL and LS-174T MUC2 K.O. Images were acquired for oblique and GFP expression on Zeiss CD7, and the area was analyzed with Imaris software. Scale bar, 200 µm. **p < 0.01; ns, not significant.

Article Snippet: After removing the blocking media, the spheroids were incubated for 45 min with primary antibody diluted in blocking media (mouse anti-human MUC2 antibody (CCP58) (Novus #NBP2-25221) diluted at 1:100 or rabbit anti-human Muc5B (Thermo Fisher Scientific, #PA5-82342) diluted at 1:25 or mouse anti-human Muc5AC (Thermo Fisher Scientific, #MA5-12178)) ( ).

Techniques: Expressing, Co-Culture Assay, Software

Journal: Frontiers in Immunology

Article Title: MUC2 expression modulates immune infiltration in colorectal cancer

doi: 10.3389/fimmu.2024.1500374

Figure Lengend Snippet: (A) Average number of Epcam+ cells recovered in the IN fraction after digestion, staining, and flow analysis without co-culture with E/A PBMCs. Significantly less Epcam+ cells were recovered for HT-29 CTRL than for HT-29 MUC2 K.O. ( p = 0.0182, paired t -test) ( N = 4 biological replicates). (B) Average number of CD45+ cells recovered in the IN fraction after digestion, staining, and flow analysis. Significantly less CD45+ cells were recovered for HT-29 CTRL than for HT-29 MUC2 K.O. ( p = 0.0433, paired t -test) ( N = 4 biological replicates). (C) Pie chart representation of average relative abundance of Epcam+ and CD45+ cells in the IN and OUT fraction after 2 days of co-culture for HT-29 CTRL and MUC2 K.O. ( N = 4 biological replicates). (D) Average number of CD45+ cells recovered in the OUT fraction after digestion, staining, and flow analysis. No significant difference in CD45+ cells recovered after co-culture with HT-29 CTRL or HT-29 MUC2 K.O. was detected despite having systematically more CD45+ cells after co-culture with HT-29 MUC2 K.O. than with HT-29 CTRL ( p = 0.1460, paired t -test) ( N = 4 biological replicates). (E) Average number of Epcam+ cells recovered in the IN fraction after digestion, staining, and flow analysis without co-culture with E/A PBMCs. No statistical difference was observed in Epcam+ cells recovered between LS-174T CTRL and LS-147T MUC2 K.O. ( p = 0.1405, paired t -test) ( N = 3 biological replicates). (F) Average number of CD45+ cells recovered in the IN fraction after digestion, staining, and flow analysis. No difference in CD45+ cells recovered was observed between LS-174T CTRL and LS-174T MUC2 K.O. ( p = 0.9202, paired t -test) ( N = 4 biological replicates). (G) Pie chart representation of average relative abundance of Epcam+ and CD45+ cells in the IN and OUT fraction after 2 days of co-culture for LS174T CTRL and MUC2 K.O. ( N = 4 biological replicates). (H) Average number of CD45+ cells recovered in the OUT fraction after digestion, staining, and flow analysis. No significant difference in CD45+ cells recovered after co-culture with LS-174T CTRL or LS-174T MUC2 K.O. was detected despite having systematically more CD45+ cells after co-culture with LS-174T CTRL than with LS-174T MUC2 K.O. ( p = 0.1262, paired t -test) ( N = 4 biological replicates). *p < 0.05; ns, not significant.

Article Snippet: After removing the blocking media, the spheroids were incubated for 45 min with primary antibody diluted in blocking media (mouse anti-human MUC2 antibody (CCP58) (Novus #NBP2-25221) diluted at 1:100 or rabbit anti-human Muc5B (Thermo Fisher Scientific, #PA5-82342) diluted at 1:25 or mouse anti-human Muc5AC (Thermo Fisher Scientific, #MA5-12178)) ( ).

Techniques: Staining, Co-Culture Assay

Journal: Frontiers in Immunology

Article Title: MUC2 expression modulates immune infiltration in colorectal cancer

doi: 10.3389/fimmu.2024.1500374

Figure Lengend Snippet: (A) Principal component analysis (PCA) based on gene expression profile in HT-29, LS-174T cultured in 2D and 3D, E/A PBMCs or IN and OUT fraction of co-culture of cancer cells with E/A PBMCs. (B) Venn diagram of DEG (FDR < 0.01, logFC >= 1) between HT-29 MUC2 K.O. + E/A PBMCs IN fraction vs. HT-29 CTRL + E/A PBMCs IN fraction ( n = 2073 genes) and HT-29 MUC2 K.O. (3D) vs. HT-29 CTRL (3D) ( n = 345). Pathway enrichment analysis was performed on genes reflecting DEG between PBMCs IN (MUC2) vs. PBMCs IN (CTRL) ( n = 1,950) by subtracting the common genes. The top 20 pathways based on p -value were selected for plotting. A comparison of those two DEG lists allows us to see that when in contact with HT-29 MUC2 K.O., E/A PBMCs increase their cell cycle and are IFN pathway activation. (C) Venn diagram of DEG (FDR < 0.01, logFC ≥ 1) between LS-174T MUC2 + E/A PBMCs IN fraction vs. LS-174T CTRL + E/A PBMCs IN fraction ( n = 891) and MUC2 K.O. (3D) vs. CNTL (3D) ( n = 463). Pathway enrichment analysis was performed on genes reflecting DEG between PBMCs IN (MUC2) vs. PBMCs IN (CTRL) ( n = 655) by subtracting the common genes. Top 20 pathways based on p -value were selected for plotting (FDR < 0.01). A comparison of those two DEG lists does not demonstrate a similar pattern with LS-174T. (D) The gene expression of other gel-forming mucin in HT-29 and LS-174T spheroids shows that LS-174T also have a strong expression of MUC5B, MUC6, and MUC19 compared to HT-29. (A, B) Histograms represent the proportion (%) of DEGs upregulated (red) or downregulated (green) in PBMCs IN (MUC2) vs. PBMCs IN (CTRL). The circles represent the pathway activation status. The blue circle indicates that the pathway is inhibited with a negative z-score, the orange circle represents that a pathway is activated with a positive z-score, the white circle represents that the pathway is neutral with zero z-score, while the gray circle indicates that the pathway activity is unknown.

Article Snippet: After removing the blocking media, the spheroids were incubated for 45 min with primary antibody diluted in blocking media (mouse anti-human MUC2 antibody (CCP58) (Novus #NBP2-25221) diluted at 1:100 or rabbit anti-human Muc5B (Thermo Fisher Scientific, #PA5-82342) diluted at 1:25 or mouse anti-human Muc5AC (Thermo Fisher Scientific, #MA5-12178)) ( ).

Techniques: Gene Expression, Cell Culture, Co-Culture Assay, Comparison, Activation Assay, Expressing, Activity Assay

Journal: Cell Reports

Article Title: Critical Role of Type III Interferon in Controlling SARS-CoV-2 Infection in Human Intestinal Epithelial Cells

doi: 10.1016/j.celrep.2020.107863

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal antibody against SARS-CoV NP (Sino biologicals MM05), mouse monoclonal against J2 (scions), mouse monoclonal against E-cadherin (BD Transductions #610181), mouse monoclonal anti-SYP (Santa Cruz Biotechnology sc-17750), and rabbit polyclonal anti-Mucin-2 (Santa Cruz Biotechnology# sc-15334) were used at 1:500 for immunofluorescence.

Techniques: Virus, Recombinant, SYBR Green Assay, RNA Extraction, cDNA Synthesis, Enzyme-linked Immunosorbent Assay, RNA Sequencing, Software

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

Journal: Translational Vision Science & Technology

Article Title: The Effect of Amniotic Membrane Transplantation or Conjunctival Autografts on the Tear Mucins MUC5A and MUC2 After Pterygium Resection: A Six-Month Follow-Up

doi: 10.1167/tvst.13.9.10

Figure Lengend Snippet: Changes in the tear levels of MUC2 and MUC5AC in pterygia and follow-up after pterygium resection. Bar graphs of tear sample quantification by ELISA for MUC2 ( A ) and MUC5AC ( B ) in patients with pterygium ( black bars ) and healthy individuals ( white bars ). The MUC2 and MUC5AC tear levels in patients with pterygium were significantly greater than those in healthy subjects. Bar graphs of MUC2 ( C ) and MUC5AC ( D ) tear level quantification by ELISA at 1, 3, and 6 months post-AMT ( black bars ) and CAG ( gray bars ) surgery. Changes in the concentrations of MUC2 and MUC5AC were significant after pterygium resection with AMT and CAG surgeries. The data are expressed as the mean ± SE ( n = 44), * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: For recognition of the mucins MUC2 and MUC5A, the plates were incubated for 2 hours at RT with the primary antibodies mouse anti-human MUC5AC (Invitrogen, Carlsbad, CA, USA) or mouse anti-human MUC2 (Abcam, Cambridge, UK).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Translational Vision Science & Technology

Article Title: The Effect of Amniotic Membrane Transplantation or Conjunctival Autografts on the Tear Mucins MUC5A and MUC2 After Pterygium Resection: A Six-Month Follow-Up

doi: 10.1167/tvst.13.9.10

Figure Lengend Snippet: The TBUT was inversely correlated with the MUC5AC and MUC2 tear levels. Spearman's correlation graph between baseline TBUT values and mucin (MUC2 and MUC5AC) tear concentrations in patients with pterygium and healthy controls; MUC2 versus TBUT ( A ); MUC5AC versus TBUT ( B ). The data are expressed as the mean ± SE ( n = 44), **** P < 0.0001.

Article Snippet: For recognition of the mucins MUC2 and MUC5A, the plates were incubated for 2 hours at RT with the primary antibodies mouse anti-human MUC5AC (Invitrogen, Carlsbad, CA, USA) or mouse anti-human MUC2 (Abcam, Cambridge, UK).

Techniques: