Journal: Nature Cell Biology

Article Title: Gasdermin E mediates resistance of pancreatic adenocarcinoma to enzymatic digestion through a YBX1–mucin pathway

doi: 10.1038/s41556-022-00857-4

Figure Lengend Snippet: a , The expression of MUC1 and MUC13 from AsPC-1 cells transfected with SGCTR or GSDME -SGs was determined by western blotting. b , AsPC-1 cells transfected with SGCTR, MUC1 -SGs, MUC13 -SGs or MUC1/MUC13 -SGs were treated with PBS or Try/Chy for 72 h. Viable cells were measured by TB staining. c , The same as b , except that AsPC-1 cells transfected with GSDME -SG, GSDME -SG/Flag- MUC1 or GSDME -SG/Flag- MUC13 were used. d , AsPC-1 or BxPC-3 cells were treated with PBS or Try/Chy for 48 h. The expression levels of GSDME, MUC1 and MUC13 were analysed by western blotting. e , The same as d , except that CCC-HPE-2, PANC-1, AsPC-1 and BxPC-3 cells were used. f , The same as b , except that AsPC-1 cells were treated with benzyl-GalNAc (2 mM), Try/Chy or Try/Chy plus benzyl-GalNAc. g , AsPC-1 cells transfected with SGCTR, MUC1 -SGs, MUC13 -SGs or MUC1/MUC13 -SGs (2.5 × 10 5 cells) were orthotopically injected into the pancreas of mice. Tumours were photographed (left) and weighed (right) ( n = 6 per group). Scale bar, 1 cm. For a – f , n = 3 biological independent experiments. P values were determined by one-way ANOVA Bonferroni’s test ( b , c , f , g ). The data represent the mean ± s.d.

Article Snippet: The cDNAs for MUC1 , MUC13 , GSDME and YBX1 were purchased from Sino-Biological (MUC1, HG12123-UT; MUC13, HG21326-UT; GSDME, HG19167-UT; YBX1, HG17046-UT).

Techniques: Expressing, Transfection, Western Blot, Staining, Injection

Journal: Nature Cell Biology

Article Title: Gasdermin E mediates resistance of pancreatic adenocarcinoma to enzymatic digestion through a YBX1–mucin pathway

doi: 10.1038/s41556-022-00857-4

Figure Lengend Snippet: a , The mRNA expression of MUCs (1, 3a, 4, 12, 13, 15 and 16) from AsPC-1 cells was determined by real-time PCR. b , The mRNA expression of MUC1 and MUC13 from SGCTR or GSDME -SGs-AsPC-1 cells was determined by qPCR. c , The expression of MUC1 and MUC13 from SGCTR or GSDME -SGs- PANC-1 cells was determined by western blot. d , The level of MUC1 and MUC13 from vector or Flag- GSDME -PANC-1 cells was determined by western blot. e The knockout efficiency of MUC1, MUC13 or MUC1/13 from AsPC-1 or BxPC-3 cells was determined by western blot. f , i , SGCTR, MUC1 -SGs, MUC13 -SGs or MUC1/13 -SGs-BxPC-3 cells (f) or SGCTR, GSDME -SG or GSDME -SG/Flag- MUC1 / 13 -AsPC-1 or PANC-1 cells (i) were treated with Trp/Chy for 72 hr. The viable cells were counted by TB staining. g , AsPC-1 cells were treated with GO-203 (5 μM), trypsin (0.5 mg/ml) /chymotrypsin (1 mg/ml) or trypsin/chymotrypsin/GO203 for 72 hr. The viable cells were counted by TB staining. h , GSDME -SG, GSDME -SG/Flag- MUC1 and GSDME -SG/Flag- MUC13 - PANC-1 cells were treated with PBS or Try/Chy for 72 hr. The viable cells were measured by TB staining. j , AsPC-1 or BxCP-3 cells were treated with PBS or Try/Chy for 48 hr. The level of GSDME, MUC1, MUC13 and β-actin was determined by analyzing the gray value on the western blot protein bands. k , AsPC-1 cells were treated with Try/Chy for 48 hr. Glycoprotein staining of MUC1 and MUC13 pulled down by immunoprecipitation assay (top). Coomassie blue staining panel represents the total amount of MUC1/13 (bottom). l , The mRNA expression of ST6GalNaC4 , ST3Gal5 , ST3Gal1 or ST3Gal2 from AsPC-1 cells treated with Trp/Chy for 24 hr. m , n , AsPC-1 or BxPC-3 cells (2.5 × 10 5 cells) were orthotopically injected into mice. After 3 days of inoculation, mice were treated with GO-203 (14 mg/kg, i.p.) once every two days for 20 days. Tumors were presented photographically (m, left) or weighed (m, right) (n = 5/group) and the mouse survival was recorded (n) (n = 6/group). Scale bar, 1 cm. o , SGCTR, GSDME -SG, GSDME -SG/Flag -Muc1/13 -1 or GSDME -SG/Flag - Muc1/13 -2 -AsPC-1 cells (2.5 × 10 5 cells) were orthotopic injected into the pancreas of NSG mice. 30 days after injection, tumors were presented photographically (left) and weighted (right) (n = 6/group). Scale bar, 1 cm. In a - j and l , n = 3 biological independent experiments. NS, no significance. NS, no significance. * P < 0.05, ** P < 0.01, *** P < 0.001, by one-way ANOVA Bonferroni’s test ( b , f-j and o ), two-tailed Student’s t-test ( a and l ) or the Log-rank survival analysis ( n ). The data represent mean ± SD.

Article Snippet: The cDNAs for MUC1 , MUC13 , GSDME and YBX1 were purchased from Sino-Biological (MUC1, HG12123-UT; MUC13, HG21326-UT; GSDME, HG19167-UT; YBX1, HG17046-UT).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Plasmid Preparation, Knock-Out, Staining, Immunoprecipitation, Injection, Two Tailed Test

Journal: Nature Cell Biology

Article Title: Gasdermin E mediates resistance of pancreatic adenocarcinoma to enzymatic digestion through a YBX1–mucin pathway

doi: 10.1038/s41556-022-00857-4

Figure Lengend Snippet: a , AsPC-1 cells transfected with SGCTR, GSDME -SG, GSDME -SG/WT- GSDME, GSDME -SG/NES- GSDME or GSDME -SG/NLS- GSDME were treated with Try/Chy for 72 h. Viable cells were measured by TB staining. b , AsPC-1 cells were treated with Try/Chy for 36 h. The cytosolic or nuclear fraction was extracted to perform an immunoprecipitation (IP) assay with anti-Flag-GSDME antibody (left). The expression of YBX1 was analysed by western blotting (right). c , AsPC-1 cells transfected with GSDME -SG/Vector or GSDME -SG/Flag-GSDME were treated with Try/Chy for 24 h. The cells were stained with anti-Flag and YBX1 antibodies and observed under a Stedycon super-resolution microscope. The plane projection of the Flag-GSDME AsPC-1 cell is indicated on the right, and the yellow spots represent colocalization of Flag-GSDME and YBX1. Scale bar, 1 μm. d , Binding ( K D ) between GSDME and YBX1 was measured by bio-layer interferometry. GSDMB was used as a negative control. e , AsPC-1 cells transfected with SGCTR, GSDME -SG, GSDME -SG/WT- GSDME , GSDME -SG/D270A or GSDME -SG/ GSDMB (2.5 × 10 5 cells) were orthotopically injected into the pancreas of NSG mice. At 30 days after injection, tumours were photographed (left) and weighed (right) ( n = 6 per group). Scale bar, 1 cm. f , AsPC-1 cells transfected with GSDME -SG/Vector, GSDME -SG/WT- GSDME , GSDME -SG/D270A, GSDME -SG/N320, GSDME -SG/N394, GSDME -SG/N419 or GSDME -SG/ GSDMB were treated with PBS or Try/Chy for 48 h. Cell viability was measured. g , The same as a , except that AsPC-1 cells transfected with SGCTR, YBX1 -SGs or YBX1 -SG/Flag- YBX1 were used. h , AsPC-1 cells transfected with SGCTR, YBX1 -SG or YBX1 -SG/Flag- MUC1/MUC13 (2.5 × 10 5 cells) were orthotopically injected into the pancreas of mice. Tumours were photographed (left) and weighed (right) ( n = 6 per group). Scale bar, 1 cm. For a – d , f and h , n = 3 biological independent experiments. P values were determined by one-way ANOVA Bonferroni’s test ( a , e – h ). The data represent the mean ± s.d.

Article Snippet: The cDNAs for MUC1 , MUC13 , GSDME and YBX1 were purchased from Sino-Biological (MUC1, HG12123-UT; MUC13, HG21326-UT; GSDME, HG19167-UT; YBX1, HG17046-UT).

Techniques: Transfection, Staining, Immunoprecipitation, Expressing, Western Blot, Plasmid Preparation, Microscopy, Binding Assay, Negative Control, Injection

Journal: Nature Cell Biology

Article Title: Gasdermin E mediates resistance of pancreatic adenocarcinoma to enzymatic digestion through a YBX1–mucin pathway

doi: 10.1038/s41556-022-00857-4

Figure Lengend Snippet: a , Immunostaining of GSDME in Trp/Chy–treated AsPC-1 and BxPC-3 cells. The mean fluorescence intensity (MFI) or fluorescence intensity of nucleus/cytosol was calculated by Image J. Scale bars, 10 μm. b , SGCTR, GSDME -SG, GSDME -SG/NES- GSDME and GSDME -SG/NLS- GSDME -PANC-1 cells were treated with Try/Chy for 72 hr. The viable cells were measured by TB staining. c , SGCTR, GSDME -SG, GSDME -SG/NES- GSDME or GSDME -SG/NLS- GSDME -AsPC-1 or PANC-1 cells were treated with Try/Chy for 48 hr. The expression of MUC1 and MUC13 were analyzed by western blot. d , Vector and Flag-GSDME-AsPC-1 cells were treated with PBS or Try/Chy for 48 hr. RNA immunoprecipitation assays were performed using an antibody against Flag and relative precipitated RNA of MUC1 (left) and MUC13 (right) levels were normalized. Antibody against IgG served as the negative control. e , Vector or Flag-GSDME-AsPC-1 cells were treated with Try/Chy for 24 hr, followed by the addition of cycloheximide (20 μM) for indicated time (0, 4, 8 or 12 hr). Cells were lysed to perform western blot analysis with an anti-Muc1 or anti-Muc13 antibody (left). The level of Muc1 (middle) or Muc13 (right) was quantified. f , GSDME -SG/Flag- GSDME -AsPC-1 cells were treated with Try/Chy for 48 hr. Cell lysates were immunoprecipitated with anti-Flag for mass spectrometry. Identified transcriptional regulatory proteins were listed. g , GSDME -SG/Vector or GSDME -SG/Flag-GSDMB-AsPC-1 cells were treated with Try/Chy for 24 hr. The cells were stained with anti-Flag and YBX1 antibodies and observed under the STEDYCON super-resolution microscope. Scale bar, 1 μm. h , Representative images of PLA in GSDME -SG/Vector, GSDME -SG/Wt- GSDME , GSDME -SG/D270A, GSDME -SG/N320, GSDME -SG/N394, GSDME -SG/N419 or GSDME -SG/ GSDMB -AsPC-1 cells were treated with PBS or Try/Chy for 48 hr. The red spots are regions of signal amplification denoting GSDME and YBX1 interaction. The PLA signals were quantified and analyzed. Scale bars, 10 μm. i , The same as (a), except that cells were stained with anti-YBX1 antibody. Scale bars, 10 μm. j , The knockout efficiency of YBX1 in AsPC-1 or PANC-1 cells was determined by western blot. k , The same as (b), except that SGCTR, YBX1 -SGs and YBX1 -SG/Flag- YBX1 - PANC-1 cells were used. l , SGCTR, YBX1 -SGs-AsPC-1 cells were treated with PBS, Try/Chy or Doxorubicin (50 μM) for 48 hr. The expression of GSDME was analyzed by Western blot. m , SGCTR or YBX1 -SGs-AsPC-1 cells (2.5 × 10 5 cells) were orthotopically injected into mice. Tumors were presented photographically (left) or weighed (right) (n = 5/group). Scale bars, 1 cm. In a - e and h , n = 3 biological independent experiments. ** P < 0.01, *** P < 0.001, by two-tailed Student’s t-test ( a , i ) or one-way ANOVA Bonferroni’s test ( b,k and m ). The data represent mean ± SD.

Article Snippet: The cDNAs for MUC1 , MUC13 , GSDME and YBX1 were purchased from Sino-Biological (MUC1, HG12123-UT; MUC13, HG21326-UT; GSDME, HG19167-UT; YBX1, HG17046-UT).

Techniques: Immunostaining, Fluorescence, Staining, Expressing, Western Blot, Plasmid Preparation, Immunoprecipitation, Negative Control, Mass Spectrometry, Microscopy, Amplification, Knock-Out, Injection, Two Tailed Test

Journal: Nature Cell Biology

Article Title: Gasdermin E mediates resistance of pancreatic adenocarcinoma to enzymatic digestion through a YBX1–mucin pathway

doi: 10.1038/s41556-022-00857-4

Figure Lengend Snippet: a , AsPC-1 cells and BxPC-3 cells transfected with SGCTR or YBX1 -SGs were treated with Try/Chy for 48 h. The expression of MUC1 and MUC13 was analysed by western blotting. b , AsPC-1 cells and BxPC-3 cells transfected with SGCTR, YBX1 -SGs, YBX1 -SG/NES- YBX1 or YBX1 -SG/NLS- YBX1 were treated with Try/Chy for 72 h. Viable cells were measured by TB staining. c , AsPC-1 cells treated with Try/Chy for 24 h were collected for ChIP–qPCR assay with anti-YBX1 and specific primers for MUC1 (left) or MUC13 (right). d , HEK-293T cells were co-transfected with MUC1 (left) or MUC13 (right) promoter luciferase reporter PGL3 and YBX1 plasmid for 24 h. Cells were then treated with Try/Chy for another 24 h, followed by analysis of luciferase activity. e , Immunostaining images (left) and quantification (right) of YBX1 from AsPC-1 cells transfected with SGCTR or GSDME -SGs and treated with Try/Chy for 36 h. Scale bar, 5 μm. f , The cell viability of AsPC-1 cells transfected with SGCTR, GSDME -SG or GSDME -SG/NLS- YBX1 was determined by TB staining. g , AsPC-1 cells transfected with SGCTR, YBX1 -SG, YBX1 -SG/NLS- YBX1 , YBX1 -SG/NES- YBX1 or YBX1 -SG/NLS-GSDME (2.5 × 10 5 cells) were injected into the pancreas of mice. Tumours were photographed (left) and weighed (right) ( n = 6 per group). Scale bar, 1 cm. For a – f , n = 3 biological independent experiments. P values were determined one-way ANOVA Bonferroni’s test ( b – g ). The data represent the mean ± s.d.

Article Snippet: The cDNAs for MUC1 , MUC13 , GSDME and YBX1 were purchased from Sino-Biological (MUC1, HG12123-UT; MUC13, HG21326-UT; GSDME, HG19167-UT; YBX1, HG17046-UT).

Techniques: Transfection, Expressing, Western Blot, Staining, Luciferase, Plasmid Preparation, Activity Assay, Immunostaining, Injection

Journal: Nature Cell Biology

Article Title: Gasdermin E mediates resistance of pancreatic adenocarcinoma to enzymatic digestion through a YBX1–mucin pathway

doi: 10.1038/s41556-022-00857-4

Figure Lengend Snippet: a , The expression of YBX1, MUC1 or MUC13 from vector or Flag-YBX1-AsPC-1 cells treated with or without Trp/Chy for 48 hr was analyzed by western blot. b , The knockout efficiency of YBX1 from BxPC-3 cells was determined by western blot. c-e , The expression of MUC1 and MUC13 from SGCTR, Flag- YBX1 , YBX1 -SG, YBX1 -SG/NLS- YBX1 or YBX1 -SG/NES- YBX1 -AsPC-1 cells stimulated by Try/Chy for 24 hr (c, d) or 48 hr (e) was determined by real-time PCR (c, d) or western blot (e). f , The log2TPM + 1 expression of GSDME in HEK239T cell line and human PDAC cell lines was quantified by RNA sequencing. g , The level of GSDME in 293 T and AsPC-1 cells were analyzed by Western blot. h , Immunostaining of GSDME from SGCTR or YBX1 -SG-AsPC-1 cells treated with Trp/Chy for 36 hr. Scale bar, 5 μm. i , Immunostaining of Flag from Flag -GSDME -AsPC-1 cells treated with Try/Chy or/and nuclear pore inhibitor WGA (100 ng/mL) for 48 hr, following SLO pretreatment, was observed under the confocal microscopy. The mean MFI of nuclear Flag was calculated by Image J. Scale bar, 10 μm. j , The cell viability from SGCTR, GSDME -SG and GSDME -SG/NLS- YBX1 -PANC-1 cells treated with or without Try/Chy was determined by TB staining. k , SGCTR, YBX1 -SG or YBX1 -SG/NLS- GSDME -AsPC-1 or BxPC-3 cells were treated with or without Try/Chy for 72 hr. The viable cells were calculated by TB staining. l , Immunostaining of Flag and Nup153 from GSDME -SG/Vector, GSDME -SG/Wt- GSDME , GSDME -SG/ GSDME -D270A, GSDME -SG/ GSDME -N320, GSDME -SG/ GSDME -N394 or GSDME -SG/ GSDME -N419- AsPC-1 cells treated with Try/Chy for 48 hr were observed under the STEDYCON super-resolution microscope. Scale bar, 1 μm. In a-k, n = 3 biological independent experiments. *** P < 0.001, by one-way ANOVA Bonferroni’s test ( c,d , h-k ). The data represent mean ± SD.

Article Snippet: The cDNAs for MUC1 , MUC13 , GSDME and YBX1 were purchased from Sino-Biological (MUC1, HG12123-UT; MUC13, HG21326-UT; GSDME, HG19167-UT; YBX1, HG17046-UT).

Techniques: Expressing, Plasmid Preparation, Western Blot, Knock-Out, Real-time Polymerase Chain Reaction, RNA Sequencing Assay, Immunostaining, Confocal Microscopy, Staining, Microscopy

Journal: Nature Cell Biology

Article Title: Gasdermin E mediates resistance of pancreatic adenocarcinoma to enzymatic digestion through a YBX1–mucin pathway

doi: 10.1038/s41556-022-00857-4

Figure Lengend Snippet: a , The expression profile of MUC1 and MUC13 from the TCGA Research Network ( http://cancergenome.nih.gov/ ). Data were presented by box plots, where the centre line shows the median, the bounds of the box show the first and third quantile, whiskers extend to the most extreme values within 1.5 interquartile range (1.5*IQR), and dots denote outliers reaching past 1.5 interquartile range. n = 179 for PDAC tissues and n = 171 for adjacent tissues. T, tumor tissues; N, adjacent normal tissues. b , c , Immunohistochemical staining of YBX1 (b) or MUC1 (c) from the pancreatic sections of PDAC patients (n = 10). Scale bar, 50 μm. **P < 0.01, ***P < 0.001, by two-tailed Mann-Whitney test ( a - c ). The data represent mean ± SD.

Article Snippet: The cDNAs for MUC1 , MUC13 , GSDME and YBX1 were purchased from Sino-Biological (MUC1, HG12123-UT; MUC13, HG21326-UT; GSDME, HG19167-UT; YBX1, HG17046-UT).

Techniques: Expressing, Immunohistochemical staining, Staining, Two Tailed Test, MANN-WHITNEY

Journal: Nature Cell Biology

Article Title: Gasdermin E mediates resistance of pancreatic adenocarcinoma to enzymatic digestion through a YBX1–mucin pathway

doi: 10.1038/s41556-022-00857-4

Figure Lengend Snippet: a , The expression profile of GSDME and YBX1 from the TCGA Research Network ( https://cancergenome.nih.gov/ ). Data are presented as box plots, where the centre line shows the median, the bounds of the box show the first and third quartiles, whiskers extend to the most extreme values within 1.5-times the interquartile range, and dots denote outliers reaching past the 1.5 interquartile range. n = 179 for PDAC tissues and n = 171 for adjacent tissues. N, normal adjacent tissue; T, tumour tissues; TPM, transcripts per million. b , c , Immunohistochemical images (left) and quantification (right) of GSDME ( b ) and MUC13 ( c ) from sections of tumour tissues and adjacent tissues of patients (P1–P3) with PDAC ( n = 10). Scale bars, 50 μm. d – f , Correlation analysis between the level of GSDME methylation ( d ), YBX1 ( e ) or MUC1 ( f ) and overall survival of patients with PDAC ( n = 177). g , Schematic of the GSDME–YBX1–mucin pathway to regulate the resistance of PDAC to digestive enzymes. P values were determined by two-tailed Mann–Whitney test ( a – c ) or two-sided Pearson’s correlation test ( d – f ). The data represent the mean ± s.d.

Article Snippet: The cDNAs for MUC1 , MUC13 , GSDME and YBX1 were purchased from Sino-Biological (MUC1, HG12123-UT; MUC13, HG21326-UT; GSDME, HG19167-UT; YBX1, HG17046-UT).

Techniques: Expressing, Immunohistochemical staining, Methylation, Two Tailed Test, MANN-WHITNEY

Journal: The Journal of Cell Biology

Article Title: BEACH domain proteins function as cargo-sorting adaptors in secretory and endocytic pathways

doi: 10.1083/jcb.202408173

Figure Lengend Snippet: Antibodies

Article Snippet: MUC13 antibody , Cell Signaling Technology , Cat# 44454.

Techniques:

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

Journal: Annals of surgical oncology

Article Title: Clinical Implications of Genetic Signatures in Appendiceal Cancer Patients with Incomplete Cytoreduction/HIPEC

doi: 10.1245/s10434-020-08841-8

Figure Lengend Snippet: Genes associated with worse overall survival in multivariable analysis in high-grade appendiceal tumors with incomplete resection, their biologic function, oncogenic evidence in other cancers, and the associations with worse overall survival in TCGA

Article Snippet: MUC13 , 1.28 , 1.01–1.60 , 0.037 , Cell signaling, mucin production , Liver, colon , None.

Techniques: Gene Expression, Chemotaxis Assay, Migration

Journal: Oncology Letters

Article Title: MUC13 -miRNA-4647 axis in colorectal cancer: Prospects to identifications of risk factors and clinical outcomes

doi: 10.3892/ol.2022.13658

Figure Lengend Snippet: Graphical representation of in silico predicted binding microRNA-4647 in the polymorphic seed sequence of MUC13 rs1532602.

Article Snippet: For MUC13 gene detection (ID Hs00217230_m1, ), cDNA was synthesized from 80 ng of total RNA in 10 μl RT reaction using TATAA GrandScript cDNA SuperMix kit (TATAA Biocenter AB), according to the manufacturer's protocol in C1000 PCR cycler (Bio-Rad Laboratories, Inc.).

Techniques: In Silico, Binding Assay, Sequencing

Journal: Oncology Letters

Article Title: MUC13 -miRNA-4647 axis in colorectal cancer: Prospects to identifications of risk factors and clinical outcomes

doi: 10.3892/ol.2022.13658

Figure Lengend Snippet: Expression of MUC13 and related miRNA in patients with colorectal cancer. (A) Expression of MUC13 in tumor and non-malignant tissue. (B) Expression of miR-4647 in tumor and non-malignant tissue (Total n=187). *P<0.05. miR, microRNA.

Article Snippet: For MUC13 gene detection (ID Hs00217230_m1, ), cDNA was synthesized from 80 ng of total RNA in 10 μl RT reaction using TATAA GrandScript cDNA SuperMix kit (TATAA Biocenter AB), according to the manufacturer's protocol in C1000 PCR cycler (Bio-Rad Laboratories, Inc.).

Techniques: Expressing

Journal: Oncology Letters

Article Title: MUC13 -miRNA-4647 axis in colorectal cancer: Prospects to identifications of risk factors and clinical outcomes

doi: 10.3892/ol.2022.13658

Figure Lengend Snippet: Expression correlation of analyzed genes. (A and B) Negative correlation of MUC13 and miR-4647 in (A) tumor tissue and (B) in non-malignant tissue (Total n=187). miR, microRNA.

Article Snippet: For MUC13 gene detection (ID Hs00217230_m1, ), cDNA was synthesized from 80 ng of total RNA in 10 μl RT reaction using TATAA GrandScript cDNA SuperMix kit (TATAA Biocenter AB), according to the manufacturer's protocol in C1000 PCR cycler (Bio-Rad Laboratories, Inc.).

Techniques: Expressing

Journal: Oncology Letters

Article Title: MUC13 -miRNA-4647 axis in colorectal cancer: Prospects to identifications of risk factors and clinical outcomes

doi: 10.3892/ol.2022.13658

Figure Lengend Snippet: Fold change difference in expression of log 2 scale of analyzed genes by non-parametric Wilcoxon test and Mann-Whitney test.

Article Snippet: For MUC13 gene detection (ID Hs00217230_m1, ), cDNA was synthesized from 80 ng of total RNA in 10 μl RT reaction using TATAA GrandScript cDNA SuperMix kit (TATAA Biocenter AB), according to the manufacturer's protocol in C1000 PCR cycler (Bio-Rad Laboratories, Inc.).

Techniques: Expressing

Journal: Oncology Letters

Article Title: MUC13 -miRNA-4647 axis in colorectal cancer: Prospects to identifications of risk factors and clinical outcomes

doi: 10.3892/ol.2022.13658

Figure Lengend Snippet: Kaplan-Meier overall survival curves stratified for high and low expression for MUC13 and miR-4647. (A) MUC13. (B) miR-4647 (Total n=187). Survival analysis was performed using the log-rank test and Kaplan-Meier plot approach. miR, microRNA.

Article Snippet: For MUC13 gene detection (ID Hs00217230_m1, ), cDNA was synthesized from 80 ng of total RNA in 10 μl RT reaction using TATAA GrandScript cDNA SuperMix kit (TATAA Biocenter AB), according to the manufacturer's protocol in C1000 PCR cycler (Bio-Rad Laboratories, Inc.).

Techniques: Expressing

Journal: Oncology Letters

Article Title: MUC13 -miRNA-4647 axis in colorectal cancer: Prospects to identifications of risk factors and clinical outcomes

doi: 10.3892/ol.2022.13658

Figure Lengend Snippet: Kaplan-Meier overall survival curves stratified for rs1532602 in MUC13 in CRC patients with different genotypes. (A) Homozygous GG genotype (P=0.04), (B) heterozygous GA genotype, (C) homozygous variant AA genotype. Survival analysis was performed using the log-rank test and Kaplan-Meier plot approach.

Article Snippet: For MUC13 gene detection (ID Hs00217230_m1, ), cDNA was synthesized from 80 ng of total RNA in 10 μl RT reaction using TATAA GrandScript cDNA SuperMix kit (TATAA Biocenter AB), according to the manufacturer's protocol in C1000 PCR cycler (Bio-Rad Laboratories, Inc.).

Techniques: Variant Assay

Journal: Oncology Letters

Article Title: MUC13 -miRNA-4647 axis in colorectal cancer: Prospects to identifications of risk factors and clinical outcomes

doi: 10.3892/ol.2022.13658

Figure Lengend Snippet: Immunohistochemical staining of MUC13 in different parts of colon. (A) Non-malignant ileum, (B) non-malignant colon, (C) non-malignant rectum, (D) cytoplasm of cancer cells and (E) endothelial cancer cells. (F) Figure from Ellipse software of cancer cells using a point-counting method where yellow points stand for positivity for mucin 13, green points for negative.

Article Snippet: For MUC13 gene detection (ID Hs00217230_m1, ), cDNA was synthesized from 80 ng of total RNA in 10 μl RT reaction using TATAA GrandScript cDNA SuperMix kit (TATAA Biocenter AB), according to the manufacturer's protocol in C1000 PCR cycler (Bio-Rad Laboratories, Inc.).

Techniques: Immunohistochemical staining, Staining, Software

Journal: Oncology Letters

Article Title: MUC13 -miRNA-4647 axis in colorectal cancer: Prospects to identifications of risk factors and clinical outcomes

doi: 10.3892/ol.2022.13658

Figure Lengend Snippet: Determination of MUC13 genotype status in colorectal cancer cell lines. HCT-116 (H line) and DLD-1 (D line) evinced heterozygous GA genotype for MUC13 rs1532602.

Article Snippet: For MUC13 gene detection (ID Hs00217230_m1, ), cDNA was synthesized from 80 ng of total RNA in 10 μl RT reaction using TATAA GrandScript cDNA SuperMix kit (TATAA Biocenter AB), according to the manufacturer's protocol in C1000 PCR cycler (Bio-Rad Laboratories, Inc.).

Techniques:

Journal: Oncology Letters

Article Title: MUC13 -miRNA-4647 axis in colorectal cancer: Prospects to identifications of risk factors and clinical outcomes

doi: 10.3892/ol.2022.13658

Figure Lengend Snippet: Transfection efficacy. (A and B) Transfection efficacy by miR-4647 mimics in (A) DLD1 and (B) HCT-116 cells. (C) Western blotting of silenced MUC13 by miR-4647. All presented results are average of 3 independent experiments (Man-Whitney test). *P<0.05 and **P<0.01. miR, microRNA; NC, negative control.

Article Snippet: For MUC13 gene detection (ID Hs00217230_m1, ), cDNA was synthesized from 80 ng of total RNA in 10 μl RT reaction using TATAA GrandScript cDNA SuperMix kit (TATAA Biocenter AB), according to the manufacturer's protocol in C1000 PCR cycler (Bio-Rad Laboratories, Inc.).

Techniques: Transfection, Western Blot, Negative Control

Journal: Oncology Letters

Article Title: MUC13 -miRNA-4647 axis in colorectal cancer: Prospects to identifications of risk factors and clinical outcomes

doi: 10.3892/ol.2022.13658

Figure Lengend Snippet: Expression levels of MUC13 after overexpression of miR-4647. (A and B) Expression of MUC13 decreased after miR-4647 overexpression in (A) DLD1 and (B) HCT-116 cells. (C and D) The adenylate kinase levels after miR-4647 overexpression in (C) DLD1 and (D) HCT-116 cells. All presented results are average of 3 independent experiments (Man-Whitney test). *P<0.05 and **P<0.01. miR, microRNA; NC, negative control.

Article Snippet: For MUC13 gene detection (ID Hs00217230_m1, ), cDNA was synthesized from 80 ng of total RNA in 10 μl RT reaction using TATAA GrandScript cDNA SuperMix kit (TATAA Biocenter AB), according to the manufacturer's protocol in C1000 PCR cycler (Bio-Rad Laboratories, Inc.).

Techniques: Expressing, Over Expression, Negative Control

Journal: Oncology Letters

Article Title: MUC13 -miRNA-4647 axis in colorectal cancer: Prospects to identifications of risk factors and clinical outcomes

doi: 10.3892/ol.2022.13658

Figure Lengend Snippet: Effect of silenced MUC13 on HCT-116 cell proliferation and migration. (A) The control expression of MUC13 after silencing. (B) The western blot analysis of silenced MUC13. (C) The effect of MUC13 silencing on the proliferation of CRC cells. (D) Migratory behaviour of CRC cells after MUC13 silencing. All presented results are average of 3 independent experiments (Man-Whitney test). *P<0.05 and ***P<0.001. NC, negative control; si-, small interfering.

Article Snippet: For MUC13 gene detection (ID Hs00217230_m1, ), cDNA was synthesized from 80 ng of total RNA in 10 μl RT reaction using TATAA GrandScript cDNA SuperMix kit (TATAA Biocenter AB), according to the manufacturer's protocol in C1000 PCR cycler (Bio-Rad Laboratories, Inc.).

Techniques: Migration, Control, Expressing, Western Blot, Negative Control