Journal: Scientific reports

Article Title: Robust and reproducible human intestinal organoid-derived monolayer model for analyzing drug absorption.

doi: 10.1038/s41598-025-95823-z

Figure Lengend Snippet: Fig. 6. Characterization of the novel human duodenal organoid-derived monolayer model. (a) Immunofluorescence analysis for E-cadherin (red), ZO-1 (green), (b) MUC2 (red), and nucleus (blue). Scale bars, 100 µm. (c) Immunofluorescence analysis depicting cross sections of the monolayer model; ZO-1 (green), nucleus (blue), and F-actin (white). Scale bars, 100 µm. Black arrows indicate the Transwell membrane. (d) and (e) Transmission electron microscopic analysis of the monolayer model. Mv, microvilli; Tj, tight junction; Sg, secretory granule-like structures. Scale bars, 10 µm for low magnification images and 2 µm for high magnification images. (f) Efflux transporter P-gp assay using the P-gp substrate digoxin with or without zosuquidar as an inhibitor. AtoB, apical to basal; BtoA, basal to apical. (g) Efflux transporter assay using BCRP substrate sulfasalazine with or without Ko143. (h) UDP-glucuronosyltransferase (UGT) functional assay using raloxifene as a UGT substrate. (i) Carboxylesterase 2 (CES2) functional assay using irinotecan as a substrate with or without loperamide as an inhibitor. The amount of SN-38, a metabolite of irinotecan, was detected in the cells of the monolayer model. (j) and (k) Midazolam permeability and metabolism assay using 2 µM midazolam as a substrate of CYP3A with or without 1-aminobenzotriazole (ABT) as a pan-CYP inhibitor. BLQ, below limit of quantification. (l) Relative gene expression determined by quantitative RT-PCR of the monolayer model with induction of VD3 or VD3 + rifampicin. Cells were treated with inducers for the last two days of the differentiation process. The P value was determined by a two-tailed unpaired t-test.

Article Snippet: The monolayer was incubated with primary antibodies [anti-ZO-1 antibody Alexa Fluor 488 (MA3-39,100-A488; Thermo Fisher Scientific), anti-E-cadherin antibody (PA5-32,178; Thermo Fisher Scientific), and anti-MUC2 antibody (NBP225,221; Novus Biologicals)] in blocking solution containing 0.3% Triton X-100 at 4 °C overnight.

Techniques: Derivative Assay, Immunofluorescence, Membrane, Transmission Assay, Functional Assay, Permeability, Gene Expression, Quantitative RT-PCR, Two Tailed Test

Journal: Communications Biology

Article Title: Human placenta mesenchymal stromal cells alleviate intestinal inflammation and repair intestinal barrier function by activating AMPK-FXR pathway

doi: 10.1038/s42003-025-08261-y

Figure Lengend Snippet: A Crypts were extracted from biopsies or tissues removed by surgery and intestinal organoids were cultured. B Representative images of cultured intestinal organoids (10×, scale bar, 100 µm). C Representative immunofluorescence images of CK20, MUC2, Caspase 3, lysozyme, PCNA, and Chromogranin A of organoids (20×, scale bar, 100 µm). D Representative bright field images (4×, scale bar, 100 µm) and the size of intestinal Orgs health and Orgs CD (at least 25 organoids from three patients and three health controls respectively in each group). E In the co-culture system, Orgs CD treated with TNF- α were cultured in the transwell and hPMSCs were cultured in the lower level. F Representative bright field images (4×, scale bar, 50 µm) of intestinal Orgs CD treated with TNF- α , and Orgs CD treated with TNF- α co-cultured with hPMSCs for 24 and 48 h. G Orgs Health and Orgs CD cultured for 5 days were treated with TNF- α , or TNF- α + hPMSCs for 48 h, and FITC-dextran 4 (FD4) flux across the organoid epitheliums normalized to the surface area of organoids was measured (40×, scale bar, 50 µm). Orgs health were used as the control (a total of 25 organoids were calculated from three independent experiments in each group). H Gene expression levels of CLDN1 and CLDN2 (five independent experiments). I WB of CLDN1 and CLDN2 from organoids of each group (experiment performed in triplicate). J Representative immunofluorescence for CLDN1 and CLDN2 (20×, scale bar, 100 µm). Five organoids were calculated in each group. Results were presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Co-culture with hPMSCs promoted Orgs CD proliferation and restored Orgs CD barrier functions disrupted by TNF- α .

Article Snippet: The fluorescence staining of organoids was performed using anti-PCNA primary monoclonal antibody (dilution, 1:200; PC10, Abcam), anti-Caspase 3 primary monoclonal antibody (dilution, 1:250; 74T2, Thermo Scientific), anti-CK20 primary monoclonal antibody (dilution, 1:200; EPR1622Y, Abcam), anti-MUC2 primary monoclonal antibody (dilution, 1:50; Ccp58, Santa Cruz), anti-lysozyme monoclonal primary antibody (dilution, 1:250; EPR2994(2), Abcam) and anti-Chromogranin A polyclonal primary antibody (dilution, 1:200; RM1025, Abcam).

Techniques: Cell Culture, Immunofluorescence, Co-Culture Assay, Control, Gene Expression

Journal: bioRxiv

Article Title: MUC2 Expression Modulates Immune Infiltration in Colorectal Cancer

doi: 10.1101/2024.08.06.594842

Figure Lengend Snippet: Relative localization and orientation of the 5 gRNAs design for Knock Out of MUC2 on Exon 2 and the primers used for PCR and sanger sequencing.

Article Snippet: MUC2 antibody (CCP58) (Novus #NBP2-25221) and HRP-conjugated β actin monoclonal antibody (Proteintech, #HRP-60008) were used as primary antibody as well as Anti-mouse IgG (H+L) antibody, human serum adsorbed and peroxidase-labeled (Seracare, # 5450-0011) as secondary anti body.

Techniques: Knock-Out, Sequencing

Journal: bioRxiv

Article Title: MUC2 Expression Modulates Immune Infiltration in Colorectal Cancer

doi: 10.1101/2024.08.06.594842

Figure Lengend Snippet: Volcano plot of the DEG using limma between mucinous versus all other histological classifications in hypermutated samples of the AC-ICAM cohort. The most significant gene expressed in mucinous samples with the higher fold change is MUC2 (p = 5.38e-16, logFC = 3.50). Two other mucins are identified as upregulated in mucinous samples: MUC5B (p = 5.40e-11, logFC = 2.50) and MUC6 (p = 2.80e-9, logFC = 2.63). Red dotes represents genes with p-value < 0.005 and grey dotes for genes with p-value > 0.005.

Article Snippet: MUC2 antibody (CCP58) (Novus #NBP2-25221) and HRP-conjugated β actin monoclonal antibody (Proteintech, #HRP-60008) were used as primary antibody as well as Anti-mouse IgG (H+L) antibody, human serum adsorbed and peroxidase-labeled (Seracare, # 5450-0011) as secondary anti body.

Techniques:

Journal: bioRxiv

Article Title: MUC2 Expression Modulates Immune Infiltration in Colorectal Cancer

doi: 10.1101/2024.08.06.594842

Figure Lengend Snippet: A. Westernblot of full length MUC2 protein (540kDa) and beta actin (housekeeping gene, 43kDa) in 3 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 3 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 3 cell line 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. Westernblot of full length MUC2 protein (540kDa) and beta actin (43 kDa) in 3 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 3 cells lines grown in 2D. MUC2 is present in abundance in LS-174T, with 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 3 cell line 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.

Article Snippet: MUC2 antibody (CCP58) (Novus #NBP2-25221) and HRP-conjugated β actin monoclonal antibody (Proteintech, #HRP-60008) were used as primary antibody as well as Anti-mouse IgG (H+L) antibody, human serum adsorbed and peroxidase-labeled (Seracare, # 5450-0011) as secondary anti body.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: bioRxiv

Article Title: MUC2 Expression Modulates Immune Infiltration in Colorectal Cancer

doi: 10.1101/2024.08.06.594842

Figure Lengend Snippet: A. Westernblot of full length MUC2 protein (540kDa) and beta actin (43 kDa) in 3 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. Westernblot of full length MUC2 protein (540kDa) and beta actin (43 kDa) in 3 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 of 100µm.

Article Snippet: MUC2 antibody (CCP58) (Novus #NBP2-25221) and HRP-conjugated β actin monoclonal antibody (Proteintech, #HRP-60008) were used as primary antibody as well as Anti-mouse IgG (H+L) antibody, human serum adsorbed and peroxidase-labeled (Seracare, # 5450-0011) as secondary anti body.

Techniques: Immunostaining

Journal: bioRxiv

Article Title: MUC2 Expression Modulates Immune Infiltration in Colorectal Cancer

doi: 10.1101/2024.08.06.594842

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 replicate each). HT-29 CTRL grow 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 replicate 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 replicate 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 zeiss CD7 fluorescent microscope of GFP and oblique of spheroids of HT-29 and LS-174T CTRL or MUC2 K.O. Scale of 200µm. 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 more round than LS-174T CTRL (unpaired t-test).

Article Snippet: MUC2 antibody (CCP58) (Novus #NBP2-25221) and HRP-conjugated β actin monoclonal antibody (Proteintech, #HRP-60008) were used as primary antibody as well as Anti-mouse IgG (H+L) antibody, human serum adsorbed and peroxidase-labeled (Seracare, # 5450-0011) as secondary anti body.

Techniques: Microscopy, Software

Journal: bioRxiv

Article Title: MUC2 Expression Modulates Immune Infiltration in Colorectal Cancer

doi: 10.1101/2024.08.06.594842

Figure Lengend Snippet: A. Surface area analysis of HT-29 CTRL and HT-29 MUC2 K.O. spheroids on day 5 (N=12 biological replicates). B. Surface area analysis of LS-174T CTRL and HT-29 MUC2 K.O. spheroids on day 5 (N=12 biological replicates).

Article Snippet: MUC2 antibody (CCP58) (Novus #NBP2-25221) and HRP-conjugated β actin monoclonal antibody (Proteintech, #HRP-60008) were used as primary antibody as well as Anti-mouse IgG (H+L) antibody, human serum adsorbed and peroxidase-labeled (Seracare, # 5450-0011) as secondary anti body.

Techniques:

Journal: bioRxiv

Article Title: MUC2 Expression Modulates Immune Infiltration in Colorectal Cancer

doi: 10.1101/2024.08.06.594842

Figure Lengend Snippet: A. Box plot of adherence junction enrichment score (ES) in HT-29 and LS-174T. The ES is up-regulated in HT-29 MUC2 K.O. compared to CTRL (p=0.041) (student t-test). In contrast, the ES is down-regulated in LS-174T MUC2 K.O. compared to CTRL (p=0.047) (student t-test). B. Representative capillary western blot (CWB) results of E-cadherin, N-cadherin and Vimentin expression in human fetal fibroblast (HFF), Hela, HT-29 CTRL and HT-29 MUC2 K.O. (N=2 technical replicates). C. Representative capillary western blot (CWB) results of E-cadherin, N-cadherin and Vimentin expression in HFF, Hela, LS-174T CTRL and LS-174T MUC2 K.O. (N=2 technical replicates). D. Quantification of band intensity of E-cadherin, N-cadherin and Vimentin normalized to beta actin expression in HFF, Hela, HT-29 CTRL and HT-29 MUC2. (N=2, technical replicates). E. Quantification of band intensity of E-cadherin, N-cadherin and Vimentin normalized to beta actin expression in HFF, Hela, LS-174T CTRL and LS-174T MUC2 K.O.. (N=2, technical replicates).

Article Snippet: MUC2 antibody (CCP58) (Novus #NBP2-25221) and HRP-conjugated β actin monoclonal antibody (Proteintech, #HRP-60008) were used as primary antibody as well as Anti-mouse IgG (H+L) antibody, human serum adsorbed and peroxidase-labeled (Seracare, # 5450-0011) as secondary anti body.

Techniques: Western Blot, Expressing

Journal: bioRxiv

Article Title: MUC2 Expression Modulates Immune Infiltration in Colorectal Cancer

doi: 10.1101/2024.08.06.594842

Figure Lengend Snippet: A. Representative micrographs of one of the individual experiments at time points 0, 24, 48 and 72 h in HT-29 CTRL and HT-29 MUC2 K.O. No cell motility toward the gap area was seen even after 72h, despite a clear cell multiplication. B. Size of scratch in HT-29 CTRL and MUC2 K.O. relative to 0 hour (N=12). C. Representative micrographs of one of the individual experiments at time points 0, 24, 48 and 72 h in LS-174T CTRL and LS-174T MUC2 K.O. No cell motility toward the gap area was seen even after 72h, despite a clear cell multiplication. D. Size of scratch in LS-174T CTRL and MUC2 K.O. relative to 0 hour (N=16).

Article Snippet: MUC2 antibody (CCP58) (Novus #NBP2-25221) and HRP-conjugated β actin monoclonal antibody (Proteintech, #HRP-60008) were used as primary antibody as well as Anti-mouse IgG (H+L) antibody, human serum adsorbed and peroxidase-labeled (Seracare, # 5450-0011) as secondary anti body.

Techniques:

Journal: bioRxiv

Article Title: MUC2 Expression Modulates Immune Infiltration in Colorectal Cancer

doi: 10.1101/2024.08.06.594842

Figure Lengend Snippet: Comparison of epithelial mesenchymal transition enrichment score between CTRL (2D) and MUC2 K.O. (2D), and between CTRL (3D) and MUC2 K.O. (3D) in A. HT-29, and B. LS-174T. P-value from student t-test.

Article Snippet: MUC2 antibody (CCP58) (Novus #NBP2-25221) and HRP-conjugated β actin monoclonal antibody (Proteintech, #HRP-60008) were used as primary antibody as well as Anti-mouse IgG (H+L) antibody, human serum adsorbed and peroxidase-labeled (Seracare, # 5450-0011) as secondary anti body.

Techniques: Comparison

Journal: bioRxiv

Article Title: MUC2 Expression Modulates Immune Infiltration in Colorectal Cancer

doi: 10.1101/2024.08.06.594842

Figure Lengend Snippet: A. HT-29 CTRL and MUC2 K.O. growth and death monitored by GFP expression over 7 days in 2D set up. Top panel shows the GFP reading for HT-29 CTRL only with 0 to 10.000 E/A PBMCs added on day 2 the following panel represents the comparison of HT-29 CTRL and HT-29 MUC2 K.O. with a defined number of E/A PBCMs. Data are presented as relative to the same cell line without co-culture with PBMCs (N=18, 3 biological replicates of 6 technical replicates). B. LS-174T CTRL and MUC2 K.O. growth and death monitored by GFP expression over 7 days in 2D set up. Top panel shows the GFP reading for LS-174T CTRL only with 0 to 10.000 E/A PBMCs added on day 2 the following panel represents the comparison of LS-174T CTRL and LS-174T MUC2 K.O. with a defined number of E/A PBCMs. Data are presented as relative to the same cell line without co-culture with PBMCs (N=18, 3 biological replicates of 6 technical replicates). C. HT-29 CTRL and MUC2 K.O. growth and death monitored by GFP expression over 9 days in 3D set up. Top panel shows the GFP reading for HT-29 CTRL only with 0 to 10.000 E/A PBMCs added on day 5 the following panel represents the comparison of HT-29 CTRL and HT-29 MUC2 K.O. with a defined number of E/A PBCMs. Data are presented as relative to the same cell line without co-culture with PBMCs (N=18, 3 biological replicates of 6 technical replicates). D. LS-174T CTRL and MUC2 K.O. growth and death monitored by GFP expression over 7 days in 3D set up. Top panel shows the GFP reading for LS-174T CTRL only with 0 to 10.000 E/A PBMCs added on day 5 the following panel represents the comparison of LS-174T CTRL and LS-174T MUC2 K.O. with a defined number of E/A PBCMs. Data are presented as relative to the same cell line without co-culture with PBMCs (N=18, 3 biological replicates of 6 technical replicates). E. Comparison of HT-29 with LS-174T CTRL or MUC2 K.O. with various number of E/A PBMCs in 2D. Data are presented as relative to the same cell line without co-culture with PBMCs (0 E/A PBMCs) (N=18, 3 biological replicates of 6 technical replicates). F. Comparison of HT-29 with LS-174T CTRL or MUC2 K.O. with various number of E/A PBMCs in 3D. Data are presented as relative to the same cell line without co-culture with PBMCs (0 E/A PBMCs) (N=18, 3 biological replicates of 6 technical replicates).

Article Snippet: MUC2 antibody (CCP58) (Novus #NBP2-25221) and HRP-conjugated β actin monoclonal antibody (Proteintech, #HRP-60008) were used as primary antibody as well as Anti-mouse IgG (H+L) antibody, human serum adsorbed and peroxidase-labeled (Seracare, # 5450-0011) as secondary anti body.

Techniques: Expressing, Comparison, Co-Culture Assay

Journal: bioRxiv

Article Title: MUC2 Expression Modulates Immune Infiltration in Colorectal Cancer

doi: 10.1101/2024.08.06.594842

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 1000 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, 3 biological replicates of 6 technical replicates). Significant differences between HT-29 and LS-174T cell lines were calculated at day 7 in 2D and 3D by student 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 area of IN fraction after rinse without co-culture. After co-culture with 2000 or 4000 PBMCs area of IN fraction spheroids is significantly smaller in HT-29 MUC2 K.O. than in HT-29 CTRL (N=12, 2 biological of 6 technical replicates) (unpaired t-test). C. Representative images of IN fraction spheroids post rinse after co-culture with 4000 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 area analyzed with Imaris software. Scale bar of 200µm. D. LS-174T 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 area of IN fraction after rinse without co-culture. No significant difference was observed between LS-174T CTRL and MUC2 K.O. at any number of E/A PBMCs co-culture (N=12, 2 biological of 6 technical replicates) (unpaired t-test). E. Representative images of IN fraction spheroids post rinse after co-culture with 4000 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 area analyzed with Imaris software. Scale bar of 200µm.

Article Snippet: MUC2 antibody (CCP58) (Novus #NBP2-25221) and HRP-conjugated β actin monoclonal antibody (Proteintech, #HRP-60008) were used as primary antibody as well as Anti-mouse IgG (H+L) antibody, human serum adsorbed and peroxidase-labeled (Seracare, # 5450-0011) as secondary anti body.

Techniques: Expressing, Co-Culture Assay, Software

Journal: bioRxiv

Article Title: MUC2 Expression Modulates Immune Infiltration in Colorectal Cancer

doi: 10.1101/2024.08.06.594842

Figure Lengend Snippet: Representative video of HT-29 CTRL, HT-29 MUC2 K.O., LS-174T CTRL, LS-174T MUC2 K.O. from day 5 to day 7 with and without E/A PBMCs.

Article Snippet: MUC2 antibody (CCP58) (Novus #NBP2-25221) and HRP-conjugated β actin monoclonal antibody (Proteintech, #HRP-60008) were used as primary antibody as well as Anti-mouse IgG (H+L) antibody, human serum adsorbed and peroxidase-labeled (Seracare, # 5450-0011) as secondary anti body.

Techniques:

Journal: bioRxiv

Article Title: MUC2 Expression Modulates Immune Infiltration in Colorectal Cancer

doi: 10.1101/2024.08.06.594842

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. 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). D. 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). 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. 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). H. 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).

Article Snippet: MUC2 antibody (CCP58) (Novus #NBP2-25221) and HRP-conjugated β actin monoclonal antibody (Proteintech, #HRP-60008) were used as primary antibody as well as Anti-mouse IgG (H+L) antibody, human serum adsorbed and peroxidase-labeled (Seracare, # 5450-0011) as secondary anti body.

Techniques: Staining, Co-Culture Assay

Journal: bioRxiv

Article Title: MUC2 Expression Modulates Immune Infiltration in Colorectal Cancer

doi: 10.1101/2024.08.06.594842

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). Pathways enrichment analysis was performed on genes reflecting DEG between PBMCs IN (MUC2) vs. PBMCs IN (CTRL) (n = 1950) by subtracting the common genes. Top 20 pathways based on p-value were selected for plotting. 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). Pathways enrichment analysis was performed on genes reflecting DEG between PBMCs IN (MUC2) vs. PBMCs IN (CTRL) (n = 655) by subtracting common the common genes. Top 20 pathways based on p-value were selected for plotting (FDR < 0.01). Comparison of those two DEG lists does not demonstrate similar pattern with LS-174T. D. Genes expression of other gel forming mucin in HT-29 and LS-174T spheroids shows that LS-174T also have strong expression of MUC5B, MUC6 and MUC19 compared to HT-29. A and 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. Blue circle indicates the pathway is inhibited with a negative z-score, orange circle represents a pathway is activated with a positive z-score, the white circle represents the pathway is neutral with zero z-score, while a gray circle indicates that the pathway activity is unknown.

Article Snippet: MUC2 antibody (CCP58) (Novus #NBP2-25221) and HRP-conjugated β actin monoclonal antibody (Proteintech, #HRP-60008) were used as primary antibody as well as Anti-mouse IgG (H+L) antibody, human serum adsorbed and peroxidase-labeled (Seracare, # 5450-0011) as secondary anti body.

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

Journal: bioRxiv

Article Title: MUC2 Expression Modulates Immune Infiltration in Colorectal Cancer

doi: 10.1101/2024.08.06.594842

Figure Lengend Snippet: IPA pathways enrichment analysis. Top 20 significant pathways based on p-value associated with DEG (FDR < 0.01, logFC >=1) between MUC2 K.O. (2D) and CTRL (2D) in A. HT-29 (n = 557) C. LS-174T (n = 1636). Between MUC2 K.O (3D) and CTRL (3D) in B. HT-29 (n = 345) D. LS-174T (n = 463). Histograms represent the proportion (%) of DEGs upregulated (red) or downregulated (green) in MUC2 K.O versus CTRL. The circles represent the pathway activation status. Blue circle indicates the pathway is inhibited with a negative z-score, orange circle represents a pathway is activated with a positive z-score, the white circle represents the pathway is neutral with zero z-score, while a gray circle indicates that the pathway activity is unknown.

Article Snippet: MUC2 antibody (CCP58) (Novus #NBP2-25221) and HRP-conjugated β actin monoclonal antibody (Proteintech, #HRP-60008) were used as primary antibody as well as Anti-mouse IgG (H+L) antibody, human serum adsorbed and peroxidase-labeled (Seracare, # 5450-0011) as secondary anti body.

Techniques: Activation Assay, Activity Assay

Journal: bioRxiv

Article Title: MUC2 Expression Modulates Immune Infiltration in Colorectal Cancer

doi: 10.1101/2024.08.06.594842

Figure Lengend Snippet: Comparison of ICR score mean value between CTRL + E/A PBMCs IN fraction and MUC2 K.O. + E/A PBMCs IN fraction in HT-29 and LS-174T. P-value from student t-test.

Article Snippet: MUC2 antibody (CCP58) (Novus #NBP2-25221) and HRP-conjugated β actin monoclonal antibody (Proteintech, #HRP-60008) were used as primary antibody as well as Anti-mouse IgG (H+L) antibody, human serum adsorbed and peroxidase-labeled (Seracare, # 5450-0011) as secondary anti body.

Techniques: Comparison

Journal: bioRxiv

Article Title: MUC2 Expression Modulates Immune Infiltration in Colorectal Cancer

doi: 10.1101/2024.08.06.594842

Figure Lengend Snippet: Comparison of genes expression of other gel forming mucin in HT-29 and LS-174T spheroids between MUC2 K.O. (in blue) and CTRL (in black) (3D). Statistical analysis represented as p-values from student’s t-test.

Article Snippet: MUC2 antibody (CCP58) (Novus #NBP2-25221) and HRP-conjugated β actin monoclonal antibody (Proteintech, #HRP-60008) were used as primary antibody as well as Anti-mouse IgG (H+L) antibody, human serum adsorbed and peroxidase-labeled (Seracare, # 5450-0011) as secondary anti body.

Techniques: Comparison, Expressing