muc13 sequence  (Thermo Fisher)

Journal: bioRxiv

Article Title: MUC13 negatively regulates tight junction proteins and intestinal epithelial barrier integrity via Protein Kinase C

doi: 10.1101/2022.10.27.513982

Figure Lengend Snippet: ( A-B ) Single-cell RNA-sequencing data of adult donors showing expression levels of mucin genes along each section of the intestinal tract ( A ) and by different cell types ( B ). ( C ) Immunofluorescence microscopy of HRT18 and Caco-2 intestinal cells stained for MUC13 cytoplasmic tail (MUC13-CT) (green) and nuclei (white). ( D ) Immunofluorescence microscopy of HRT18 cells with antibodies against MUC13-CT and occludin, in combination with DAPI from basal to lateral Z planes. ( E ) Immunofluorescence microscopy of HRT18 cells with monoclonal MUC13 antibody against the extracellular domain. White scale bars represent 20 mM.

Article Snippet: The optimized MUC13 sequence was ordered from Thermo Fisher.

Techniques: RNA Sequencing Assay, Expressing, Immunofluorescence, Microscopy, Staining

Journal: bioRxiv

Article Title: MUC13 negatively regulates tight junction proteins and intestinal epithelial barrier integrity via Protein Kinase C

doi: 10.1101/2022.10.27.513982

Figure Lengend Snippet: ( A ) Immunofluorescence of HRT18 intestinal cells stained for MUC13 cytoplasmic tail (MUC13-CT) (green), E-cadherin (red), β-actin (blue), and DAPI (white). White scale bars represent 20 mM. Pictures were taken at different heights in the epithelial monolayer (Z).

Article Snippet: The optimized MUC13 sequence was ordered from Thermo Fisher.

Techniques: Immunofluorescence, Staining

Journal: bioRxiv

Article Title: MUC13 negatively regulates tight junction proteins and intestinal epithelial barrier integrity via Protein Kinase C

doi: 10.1101/2022.10.27.513982

Figure Lengend Snippet: ( A ) CRISPR/Cas9 targeting strategy using two guide RNAs directed against exon 2 or exon 10 of MUC13. ( B ) Schematic representation of WT and MUC13-ΔCT MUC13 domain structure. ( C ) Wild type MUC13 protein sequence with domains color-coded as in . ( D ) Confirmation PCR of WT and ΔMUC13 cell lines (left), and WT and MUC13-ΔCT cell lines (right). ( E ) Immunoblot of WT, ΔMUC13, and MUC13-ΔCT cell lines with anti-MUC13-CT antibody and actin loading control. Molecular mass standards (kDa) are indicated on the left. ( F) Immunofluorescence confocal image of WT, ΔMUC13, and MUC13-ΔCT cells stained for MUC13-CT (green) and nuclei (white). White scale bars represent 20 mM. ( G ) Immunofluorescence confocal images of WT, ΔMUC13, and MUC13-ΔCT cells stained for MUC13-ED (green) and nuclei (white). White scale bars represent 10 mM. ( H ) Immunofluorescence confocal image of WT Ctr (empty plasmid), WT+pMUC13 (with inducible MUC13-GFP construct), ΔMUC13 Ctr, and ΔMUC13+pMUC13 complementation cell lines after doxycycline induction for 24h. MUC13-GFP is depicted in green, and nuclei are shown in white. White scale bars represent 40 mM.

Article Snippet: The optimized MUC13 sequence was ordered from Thermo Fisher.

Techniques: CRISPR, Sequencing, Western Blot, Immunofluorescence, Staining, Plasmid Preparation, Construct

Journal: bioRxiv

Article Title: MUC13 negatively regulates tight junction proteins and intestinal epithelial barrier integrity via Protein Kinase C

doi: 10.1101/2022.10.27.513982

Figure Lengend Snippet: ( A ) Immunofluorescence confocal image of WT, ΔMUC13, and MUC13-ΔCT cell monolayers showing occludin (green), E-cadherin (red), and nuclei (DAPI; white) staining. White scale bars represent 20 mM. ( B ) Transepithelial electrical resistance (TEER) buildup in cell monolayers grown for up to 14 days. ( C ) TEER buildup in 2-weeks-differentiated monolayers. ( D ) Quantification of cell nuclei per plane by confocal microscopy (DAPI) in cell monolayers after 14 days of differentiation. ( E ) TEER buildup in the MUC13 overexpression and complementation WT+pMUC13 and ΔMUC13+pMUC13 cell lines. Doxycycline was added on day 14 as indicated by an arrow. ( F ) Fold change (log2) of TEER increase in WT+pMUC13 and ΔMUC13+pMUC13 cells on day 19 compared to day 14 before the addition of doxycycline. ( G ) Paracellular passage of Lucifer Yellow CH substrate and FITC-dextran particles in 14-days-differentiated cell monolayers. ( H ) Paracellular permeability assay with LPS from Escherichia coli 0111:B4 in 14-days differentiated monolayers. ( I ) Fold change (log2) compared to time 0 of TEER increase in 14 days-differentiated WT and ΔMUC13 cell monolayers after addition of Lactobacillus plantarum (LP) for 42 h at MOI 50. ( J ) Quantification of cell nuclei per plane by confocal microscopy (DAPI) in WT and ΔMUC13 cell monolayers after 42 h incubation with LP. All graphs represent the average and SEM of three independent experiments. ns, non-significant; *, p<0.05; ** p<0.01; *** p<0.001.

Article Snippet: The optimized MUC13 sequence was ordered from Thermo Fisher.

Techniques: Immunofluorescence, Staining, Confocal Microscopy, Over Expression, Permeability, Incubation

Journal: bioRxiv

Article Title: MUC13 negatively regulates tight junction proteins and intestinal epithelial barrier integrity via Protein Kinase C

doi: 10.1101/2022.10.27.513982

Figure Lengend Snippet: ( A ) Subcellular fractionation protocol for the enrichment of the membrane fraction from epithelial monolayers. 1) Intestinal epithelial cell lines were grown for 2 weeks in 10 cm 2 culture dishes. 2) Monolayers were lysed by passing through a needle in hyperosmotic fractionation buffer. 3) Nuclei (and unbroken cells) were pelleted by centrifugation and stored as the nuclear fraction (N). 4) The supernatant was collected and centrifuged again to pellet mitochondria. 5) Supernatant was again collected, and membranes were pelleted by ultracentrifugation. 6) The supernatant containing the cytosolic fraction (C) was stored. The pellet was washed and resuspended in fractionation buffer and pelleted by ultracentrifugation a second time to increase purity. 6) The resulting pellet was resuspended in TSB + 1% SDS buffer and stored as the membrane fraction (M). ( B ) Immunoblot analysis of subcellular fractionation of two WT, ΔMUC13, and MUC13-ΔCT cell lines using Na + /K + -ATPase (membrane marker), Histone-H3 (nuclear marker), and β-actin (cytoplasmic marker). C (cytosolic fraction), M (membrane fraction), N (nuclear fraction). Molecular mass standards (kDa) are indicated on the left. ( C ) Relative abundance of cell junction proteins identified by mass spectrometry in membrane fractions of WT, ΔMUC13, and MUC13-ΔCT monolayers grown for 2 weeks.

Article Snippet: The optimized MUC13 sequence was ordered from Thermo Fisher.

Techniques: Fractionation, Centrifugation, Western Blot, Marker, Mass Spectrometry

Journal: bioRxiv

Article Title: MUC13 negatively regulates tight junction proteins and intestinal epithelial barrier integrity via Protein Kinase C

doi: 10.1101/2022.10.27.513982

Figure Lengend Snippet: ( A ) Degradation rates of biotinylated occludin, claudin-1, claudin-4, and E-cadherin analyzed by immunoblot in cell monolayers. Cells were incubated with biotin-NHS on day 0 and the presence of biotinylated proteins was determined on days 0, 1, and 3. T (total lysate), B (elution from streptavidin beads). The assay was performed at least three times and representative images are shown. Molecular mass standards (kDa) are indicated on the left. ( B ) Relative protein abundance of biotinylated occludin, claudin-1, claudin-4, and E-cadherin proteins on days 0, 1, and 3. ( C ) Immunoblot of occludin, ZO-1, claudin-1, claudin-4, E-cadherin, and β-actin in total lysates of monolayers grown for 2 weeks. The assay was performed at least three times and representative images are shown. Molecular mass standards (kDa) are indicated on the left. ( D-F ) TEER buildup of WT (D), ΔMUC13 (E), and MUC13-ΔCT (F) cell lines over time in the presence of kinase inhibitors ML-7 (MLCK), Y-27632 (ROCK), and GF-109203X (PKC). Inhibitors were added on days 3, 6, and 9 at a concentration of 50 mM (ML-7 and Y-27632) and 20 mM (GF-109203X). One representative clone for each cell line was used in these experiments. Bars represent the average and SEM of three independent experiments. *, p<0.05; ** p<0.01.

Article Snippet: The optimized MUC13 sequence was ordered from Thermo Fisher.

Techniques: Western Blot, Incubation, Concentration Assay

Journal: bioRxiv

Article Title: MUC13 negatively regulates tight junction proteins and intestinal epithelial barrier integrity via Protein Kinase C

doi: 10.1101/2022.10.27.513982

Figure Lengend Snippet: ( A ) Schematic representation of WT MUC13 domain structure (left) and protein sequence (right). The transmembrane domain (grey), the cytoplasmic tail (red), and two predicted PKC binding motifs (black boxes) are marked. (B) Immunoblot analysis of PKCα, PKCδ, and β-actin in total lysates of monolayers grown for 2 weeks. Molecular mass standards (kDa) are indicated on the left. (C) Immunoblot analysis of isolated membrane fractions from monolayers grown for 2 weeks in the presence/absence of 20 mM PKC inhibitor (GF-109203X) added every 3 days. Claudin-1, claudin-3, claudin-4, and the control protein Na + /K + -ATPase are shown. Molecular mass standards (kDa) are indicated on the left. (D) Quantification of relative protein expression of claudin-1, claudin-3, and claudin-4 in isolated fractions of cells grown in the presence/absence of GF-109203X as depicted in C. All assays were performed at least three times and representative images are shown. One representative clone for each cell line was used in these experiments. Bars represent average and SEM of three independent experiments. ns, non-significant; ** p<0.01; *** p<0.001.

Article Snippet: The optimized MUC13 sequence was ordered from Thermo Fisher.

Techniques: Sequencing, Binding Assay, Western Blot, Isolation, Expressing

Journal: bioRxiv

Article Title: MUC13 negatively regulates tight junction proteins and intestinal epithelial barrier integrity via Protein Kinase C

doi: 10.1101/2022.10.27.513982

Figure Lengend Snippet: ( A ) In wild type cells, MUC13 localizes to both the apical surface and tight junction (TJ) region of the lateral membrane. Cell junction complexes that contain claudins, occludin, ZOs, and E-cadherin, are assembled along the lateral membrane. Under normal conditions, there is some paracellular passage of ions and small solutes, a process that is controlled by the TJ proteins claudins and occludin. MUC13 cytoplasmic tail has a putative PKC binding motif, which may play are role in recruiting PKC and controlling its activity and/or stability. Cell junction proteins such as claudins, occludin, and ZO-1 also can be targeted by PKCs. ( B ) In the absence of the complete MUC13 protein, TJ proteins (occludin, claudins, and ZO-1) are accumulating at the membrane over time, causing increased transepithelial resistance (TEER) and lower paracellular passage of small solutes. The TEER buildup in ΔMUC13 cells is dependent on MLCK, ROCK and PKC kinases. The accumulation of claudins at the membrane in ΔMUC13 cells is PKC-dependent and is not caused by slower degradation rates of TJ proteins through recycling endosomes. ( C ) Removal of the MUC13 cytoplasmic tail leads to an intermediate phenotype with some accumulation of claudin-1, -3, -4, and ZO-1 at the membrane, but to a lower extent compared to the full knockout. The role of PKC in this cell line remains to be determined. MUC13-ΔCT cells are less permeable to small solutes but do not show a significant increase in TEER when compared to WT cells. The degradation rate of TJ proteins in MUC13-ΔCT cells is comparable to WT and ΔMUC13.

Article Snippet: The optimized MUC13 sequence was ordered from Thermo Fisher.

Techniques: Binding Assay, Activity Assay, Knock-Out