Journal: Nature Communications

Article Title: Polystyrene nanoplastics disrupt the intestinal microenvironment by altering bacteria-host interactions through extracellular vesicle-delivered microRNAs

doi: 10.1038/s41467-025-59884-y

Figure Lengend Snippet: A Alcian blue-periodic acid Schiff (AB-PAS) staining of intestinal mucus in mice with or without NP treatment. Scale bars indicate 100 µm (upper) and 50 µm (lower). Data were shown as mea n ± SEM ( n = 12). B IHC stain of intestinal MUC-13 in NP-treated mice. Scale bars indicate 100 µm (upper) and 50 µm (lower). Data were shown as mea n ± SEM ( n = 12) (*** p value < 0.001). C MUC13 expression in NP-treated enterocyte-like differentiated Caco-2 cells for 48 h by ICC stain (Scale bars indicate 20 µm), qPCR, and Western blot. Data are presented as means ± SD ( n = 3). Significant difference was shown by different letters (* p < 0.05; *** p < 0.001). D Western blot analysis of MUC-13 levels in goblet-like LS174T cells treated with NP for 48 h. E Heatmap predicting various intestinal miRNAs suppressing MUC-13 in NP-exposed mice. Validation of miR-700-5p interference on MUC-13 in enterocyte-like differentiated Caco-2 cells by ( F ) qPCR. G ICC stain (Scale bars indicate 20 µm), and ( H ) Western blot. Data are presented as means ± SD ( n = 3). Significant difference was shown by different letters (*** p < 0.001). I Schematic of NP im p act on MUC-13 and mucus secretion in the gut.

Article Snippet: For Western blot, the primary antibodies for beta-actin (SC-47778, Santa Cruz, Dallas, Texas, USA), MUC13 (bs-10074R, Bioss, Boston, Massachusetts, USA), GAPDH (GTX100118, GENETEX, Texas, USA), ZO-1 (ab96587, Abcam, Bristol, UK), and OCC (ab216327, Abcam, Bristol, UK) were used to evaluated protein level.

Techniques: Staining, Expressing, Western Blot, Biomarker Discovery

Journal: Nature Communications

Article Title: Polystyrene nanoplastics disrupt the intestinal microenvironment by altering bacteria-host interactions through extracellular vesicle-delivered microRNAs

doi: 10.1038/s41467-025-59884-y

Figure Lengend Snippet: A The effects of NP treatment (1 × 10 10 particles/mL) on the growth of various lactic acid bacteria ( L. paracasei, L. acidophilus , and P. acidiloctici ) , Lachnospiraceae sp. (TSD-26; ATCC), and Ruminococcaceae sp. (TSD-27; ATCC). B Schematic of experimental process by interactions between bacterial EV and cell-derived EV. C Impact of Lachnospiraceae sp.-derived EV without or with NP treatment (1 × 10 10 particles/mL) for 18 h on the growth of different bacterial species ( L. paracasei , L. acidophilus , P. acidiloctici , and Ruminococcaceae sp.). D The impact of Ruminococcaceae sp.-derived EV without or with NP treatment (1 × 10 10 particles/mL) for 44 h on the growth of different bacterial species ( L. paracasei , L. acidophilus , P. acidiloctici , and Lachnospiraceae sp.). E Impact of goblet-like LS174T cells without or with NP treatment (10 6 particles/mL) for 48 h on the growth of different bacterial species ( L. paracasei, L. acidophilus, P. acidiloctici , and Lachnospiraceae sp. and Ruminococcaceae sp.). F Western blot of MUC13 inhibition by Lachnospiraceae sp.-derived EV. Data were shown as mea n ± SD ( n = 3) (* p value < 0.05). G Schematic representation summarizing the proposed mechanisms of NP-induced modulation of gut microbiota via EV. NP are taken up by Lachnospiraceae , whose EV suppress MUC13 expression in goblet cells. Concurrently, NP-modified EV from goblet cells promote the growth of Ruminococcaceae , collectively contributing to gut microbiota imbalance and potential intestinal barrier dysfunction.

Article Snippet: For Western blot, the primary antibodies for beta-actin (SC-47778, Santa Cruz, Dallas, Texas, USA), MUC13 (bs-10074R, Bioss, Boston, Massachusetts, USA), GAPDH (GTX100118, GENETEX, Texas, USA), ZO-1 (ab96587, Abcam, Bristol, UK), and OCC (ab216327, Abcam, Bristol, UK) were used to evaluated protein level.

Techniques: Bacteria, Derivative Assay, Western Blot, Inhibition, Expressing, Modification

Journal: Gut microbes

Article Title: Detecting host responses to microbial stimulation using primary epithelial organoids.

doi: 10.1080/19490976.2023.2281012

Figure Lengend Snippet: Figure 3. Development and characterization of a 2D model for primary intestinal epithelial cells. (a-c) Detection of tight junction marker, ZO1 (red), F-actin with phalloidin (green), and nuclei, DAPI (blue) (a), enterocyte marker, ALDOB (red), and nuclei, DAPI (blue) (b) and goblet cell marker, MUC13 (red), and nuclei, DAPI (blue) (c) in confluent layers of intestinal epithelial cells. Scale bar 25 µm. Insert in (a) shows a Z-section of the cell layer with clear localization of ZO1 and F-actin at the apical surface. Scale bar 10 µm. (d) Left: Primary epithelial cells seeded in 2D form a confluent layer within 7 days from seeding. Right: MA-plot based on RNA-seq analysis of 2D vs 3D cells. Y axis shows 2D vs 3D log2FC, and X shows baseline expression in TPM (transcript per million). Color shows the number of genes in each bin. Full list of GO-terms enriched in 2D and 3D cultures in Table S2. (e) Gene set enrichment analysis of the uniquely annotated genes associated with 2D or 3D cultures versus published gene signatures representing enterocyte differentiation, general differentiation in the intestine, proliferation, and stem cells. X axis show observed vs expected overlap based on randomly selected genes. (f,g) UMAP plots of single-cell RNAseq data acquired using the 10× platform. c: Colors show cells cultured in 2D and 3D. d: Colors show nine cell clusters, defined by the Leiden method. (h) Cell distribution between the different clusters from panel j. Bars

Article Snippet: The following primary antibodies were used: anti-ZO1 (61-7300; Invitrogen), anti Muc13 (HPA045163; Atlas Antibodies), and anti-AldoB (HPA073201; Atlas Antibodies) and secondary antibodies Alexa Fluor 647 polyclonal donkey anti-rabbit.

Techniques: Marker, RNA Sequencing, Expressing, Cell Culture

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: iScience

Article Title: Bidirectional signals generated by Siglec-7 and its crucial ligand tri-sialylated T to escape of cancer cells from immune surveillance

doi: 10.1016/j.isci.2024.111139

Figure Lengend Snippet: Siglec-7 binding is enhanced by four sialyltransferases in colon cancer cell lines, and its ligand glycan is O-glycan on mucin-type proteins (A) Flow cytometry analysis of Siglec-7-Fc binding to parent DLD-1 and stably expressing cells of sialyltransferases that have been identified as Siglec-7 ligand-synthase genes ( Figure S2 A). As a result, ST3Gal1, ST6GalNAc1, ST6GalNAc3, and ST8Sia6 were identified to be capable of the induction of Siglec-7 binding. DLD-1 co-transfected with ST3Gal1 and ST6GalNAc3 genes is C3. Co-transfectant DLD-1 of ST3Gal1 and ST6GalNAc1 genes is C4. ST8Sia6 gene transfectant DLD-1 is C8. Blue-filled lines indicate negative controls, and purple lines indicate Siglec-7-Fc binding. (B and C) Effects of inhibition of N-glycosylation or O-glycosylation on the binding of Siglec-7. DLD-1 and the transfectants were treated with 20 nM kifunensine or DMSO as a negative control or 2 mM benzyl-α-GalNAc or DMSO as a negative control for 3 days, respectively. They were detached and incubated with Siglec-7-Fc (5 μg/100 μL) for 1 h and then with anti-human Fc IgG-FITC, and then analyzed using flow cytometry. (B) Kifunensine, an N-glycosylation inhibitor, showed no effect on the binding of Siglec-7. (C) Benzyl-α-GalNAc, an O-glycosylation synthesis inhibitor, led to a significant decrease in the binding of Siglec-7 in all three clones. (D) N- and O-glycan synthesis inhibition was performed on the Siglec-7-binding cell colon adenocarcinoma cell line SW837 and its effects were evaluated. (E) Biotinylated membrane proteins derived from DLD-1 and the ST transfectants were pulled down using Siglec-7-Fc or human IgG Fc only as a protein control Fc (Con-Fc) and protein A beads. (F) They were electrophoresed and transferred onto a PVDF membrane. It was used for the avidin-biotin complex detection system (ABC kit) and ECL. Specific bands were detected at 175 kDa< and 50 kDa in ST-transfectants. A specific band detected at 120 kDa in C8 was Mucin-13 (MUC13) ( Figure S2 C). (G) Immuno-blotting using anti-PODXL antibody. The upper panel indicates pull-down samples with Siglec-7-Fc or control-Fc. Lower panel indicates input lysates.

Article Snippet: The antibodies were Anti-β-Actin antibody (1:5000, Sigma), Anti-AKT (1:1000, Cell Signaling Technology), Anti- p -AKT (S473) (1:1000, Cell Signaling Technology), Anti-phospho ERK1/2 (1:1000, Cell Signaling Technology), Anti-p44/42 MAPK (ERK1/2) (1:1000, Cell Signaling Technology), Anti-rabbit IgG-HRP (1:1000, Cell Signaling Technology), Goat TrueBlot anti-goat Ig HRP (1:2000, ebioscience), Anti-Mouse IgG HRP-Linked Whole Ab (1:2000, GE HealthCare), Anti-MUC13 (1:1000, IMGENEX), Anti- p -Tyr (4G10) HEP conjugate (1:5000, Millipore), Anti-SHP-1 (1:1000, Millipore), Anti-Siglec-7/CD328 antibody (1:1000, R&D), Anti-PODXL (3D3) (1:1000, Santa Cruz), Anti- p -Tyr (PY20) HRP conjugate (1:2000, BD), Rat anti-PDPN (NZ-1) (1:1000, AngioBio Co), Anti-Rat IgG-HRP (1:2000, Cosmo bio), ABC standard kit Elite (5 μL/mL each, Vector) for immunoblotting, Anti-rabbit IgG Fab2 Alexa 555 (1:400, Cell Signaling Technology), Anti-mouse IgG Fab2 Alexa 647 (1:400, Cell Signaling Technology), Anti-mouse IgG Alexa 488 (1:400, Invitrogen), Phalloidin Alexa 647 (1:400, Invitrogen), DAPI (1:500, Sigma) for immunocyte chemistry.

Techniques: Binding Assay, Flow Cytometry, Stable Transfection, Expressing, Transfection, Cotransfection, Inhibition, Negative Control, Incubation, Clone Assay, Membrane, Derivative Assay, Control, Avidin-Biotin Assay

Journal: iScience

Article Title: Bidirectional signals generated by Siglec-7 and its crucial ligand tri-sialylated T to escape of cancer cells from immune surveillance

doi: 10.1016/j.isci.2024.111139

Figure Lengend Snippet:

Article Snippet: The antibodies were Anti-β-Actin antibody (1:5000, Sigma), Anti-AKT (1:1000, Cell Signaling Technology), Anti- p -AKT (S473) (1:1000, Cell Signaling Technology), Anti-phospho ERK1/2 (1:1000, Cell Signaling Technology), Anti-p44/42 MAPK (ERK1/2) (1:1000, Cell Signaling Technology), Anti-rabbit IgG-HRP (1:1000, Cell Signaling Technology), Goat TrueBlot anti-goat Ig HRP (1:2000, ebioscience), Anti-Mouse IgG HRP-Linked Whole Ab (1:2000, GE HealthCare), Anti-MUC13 (1:1000, IMGENEX), Anti- p -Tyr (4G10) HEP conjugate (1:5000, Millipore), Anti-SHP-1 (1:1000, Millipore), Anti-Siglec-7/CD328 antibody (1:1000, R&D), Anti-PODXL (3D3) (1:1000, Santa Cruz), Anti- p -Tyr (PY20) HRP conjugate (1:2000, BD), Rat anti-PDPN (NZ-1) (1:1000, AngioBio Co), Anti-Rat IgG-HRP (1:2000, Cosmo bio), ABC standard kit Elite (5 μL/mL each, Vector) for immunoblotting, Anti-rabbit IgG Fab2 Alexa 555 (1:400, Cell Signaling Technology), Anti-mouse IgG Fab2 Alexa 647 (1:400, Cell Signaling Technology), Anti-mouse IgG Alexa 488 (1:400, Invitrogen), Phalloidin Alexa 647 (1:400, Invitrogen), DAPI (1:500, Sigma) for immunocyte chemistry.

Techniques: Blocking Assay, Plasmid Preparation, Purification, Control, Staining, Recombinant, Membrane, Mutagenesis, Cytotoxicity Assay, Clone Assay, Expressing, Sequencing, Software

Journal: Oncotarget

Article Title: MicroRNA-145 targets MUC13 and suppresses growth and invasion of pancreatic cancer

doi:

Figure Lengend Snippet: (A) Identification of a putative miR-145-binding site in the MUC13 3′ UTR region. Seven bases (597 through 603) of the MUC13 3′ UTR are perfect matches (seed sequence) for miR-145 binding. (B) Comparison of the MUC13-binding element among mammals demonstrates a high degree of conservation. (C) MUC13 expression on miR-145 transfection was examined at protein and mRNA levels by Western blot analyses and semi-quantitative reverse transcription–PCR (RT-PCR), respectively. (D) Luciferase reporter assay was used to examine the miR-145-mediated regulation of gene expression. HPAF-II cells were transiently co-transfected for 48 h with reporter plasmids (0.5 μg, WT or MUT) and 100 nM of miR-145 or NC mimic using Lipofectamine 2000. Luciferase (Firefly; test and Renilla, transfection efficiency control) activity was assessed using a dual-luciferase assay system. Data are presented as normalized fold change in luciferase activity (mean ± SD; n= 3, * P <0.05).

Article Snippet: The mature miRNA and MUC13 expression levels were determined by real-time PCR using Taqman PCR master mixture and specific primers using Taqman expression Assay (Assay id 002278, Hs01550533_m1 respectively; Applied Biosystems).

Techniques: Binding Assay, Sequencing, Comparison, Expressing, Transfection, Western Blot, Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Luciferase, Reporter Assay, Gene Expression, Control, Activity Assay

Journal: Oncotarget

Article Title: MicroRNA-145 targets MUC13 and suppresses growth and invasion of pancreatic cancer

doi:

Figure Lengend Snippet: (A and B) Cells were transfected with miR-145 mimic, NC or miR-145 inhibitor in addition to miR-145 mimic for 48 h. Immunoblotting was performed for analysis of indicated proteins. (C and D) Confocal microscopy of HPAF-II, Capan-1 and HPAF-II sh-MUC13 cells treated with NC and miR-145 mimic. Data show a decrease in MUC13 (green) and HER2 (green) and an increase in p53 (pink) levels that reciprocated the results from the RNAi experiments using HPAF-II sh-MUC13 cells.

Article Snippet: The mature miRNA and MUC13 expression levels were determined by real-time PCR using Taqman PCR master mixture and specific primers using Taqman expression Assay (Assay id 002278, Hs01550533_m1 respectively; Applied Biosystems).

Techniques: Transfection, Western Blot, Confocal Microscopy

Journal: Oncotarget

Article Title: MicroRNA-145 targets MUC13 and suppresses growth and invasion of pancreatic cancer

doi:

Figure Lengend Snippet: AsPC-1 cells (gemcitabine resistant cells) were transfected with miR-145 mimic or NC and then treated with a gemcitabine-conditioned medium (100 nM) for 48 h followed by the (A) matrigel invasion assay. Cells were photographed and counted using an imaging system. Bars represent mean ± SD; (n=3); *p<0.01 and **p<0.001. (B) Western blotting for the analysis of expression of HER2, MUC13 and the gemcitabine target, Mcl-1. (C) Flow cytometry analysis of miR-145-transfected human pancreatic ASPC-1 and HPAF-II cells indicating an increase in the G0–G1 stage. Data are representative of one of three similar experiments.

Article Snippet: The mature miRNA and MUC13 expression levels were determined by real-time PCR using Taqman PCR master mixture and specific primers using Taqman expression Assay (Assay id 002278, Hs01550533_m1 respectively; Applied Biosystems).

Techniques: Transfection, Invasion Assay, Imaging, Western Blot, Expressing, Flow Cytometry

Journal: Oncotarget

Article Title: MicroRNA-145 targets MUC13 and suppresses growth and invasion of pancreatic cancer

doi:

Figure Lengend Snippet: The antitumor effect of miR-145 was confirmed by in vivo experiments using xenograft models. (A) The antitumor effect of miR-145 was analyzed after intratumoral administration of miR-145 in established HPAF-II tumors. Average tumor volumes were calculated. Bars represent mean ± SD; *p<0.05 and **p<0.01. (B) Also, the xenograft tumors from miR-145 treated mice were analyzed for changes in MUC13 and HER2 expression (Original magnifications 40X) through IHC and miR-145 levels (Original magnifications 10X) using in situ hybridization followed by microscopy.

Article Snippet: The mature miRNA and MUC13 expression levels were determined by real-time PCR using Taqman PCR master mixture and specific primers using Taqman expression Assay (Assay id 002278, Hs01550533_m1 respectively; Applied Biosystems).

Techniques: In Vivo, Expressing, In Situ Hybridization, Microscopy

Journal: Oncotarget

Article Title: MicroRNA-145 targets MUC13 and suppresses growth and invasion of pancreatic cancer

doi:

Figure Lengend Snippet: Immunohistochemistry and in situ hybridization was used to detect MUC13 and miR-145, respectively, on the tissue microarray slides (procured from US Biomax, Inc., Rockville, MD) in various (A) PanIN lesions (original magnifications: MUC13 60X; miR-145 20X), (B) adenocarcinoma (original magnifications 60X) and (C) adjacent normal (Adj) (original magnifications: MUC13 40X; miR-145 20X) and normal pancreatic cancer cells (original magnifications: 20X).

Article Snippet: The mature miRNA and MUC13 expression levels were determined by real-time PCR using Taqman PCR master mixture and specific primers using Taqman expression Assay (Assay id 002278, Hs01550533_m1 respectively; Applied Biosystems).

Techniques: Immunohistochemistry, In Situ Hybridization, Microarray