Journal: Molecular & Cellular Proteomics : MCP

Article Title: O -Mannose Glycosylations Influence E-Cadherin Functional Interactions

doi: 10.1016/j.mcpro.2026.101559

Figure Lengend Snippet: Mapping the O -Man dependent E-cadherin interactome using IP screening. A , Schematic diagrams and structural model of CDH1 EC domains : ( left ) CDH1 is a transmembrane protein with five EC domains that form cis- and trans interactions; ( middle ) TMTC2 mediates O -Man on CDH1 EC B-strands, while TMTC3 mediates glycosylations on G-strands ( O -Man structures were grafted onto an AlphaFold model of EC4 using the GlycoShape tool – the mannoses are depicted as green sticks and translucent surfaces on recipient serine and threonine residues ); ( right ) schematic of the β-strand arrangement of an EC domain, highlighting O -Man sites ( green dots ) on the B- ( red ) and G- ( blue ) strands of EC2-4. B , Schematic diagram of the IP-MS-based interactome screen applied to CDH1 : Cryomilled cells are distributed to a 96-well plate and combined with different extractants; CDH1-associated complexes are affinity enriched from each extract using an antibody coupled magnetic medium and then analyzed by protein MS; the compositions of the enriched macromolecular assemblies will vary according to the stabilizing/destabilizing responses of the protein constituents and a putative interactome is constituted by the combined results. C , Results of the IP screen using 32 extraction conditions : ( upper ) silver-stained SDS-PAGE gel showing CDH1 capture by IP screening; ( lower ) hierarchical clustering of MS data, with log 2 -transformed protein abundance values from Proteome Discoverer displayed by color. Grey shading in the heatmap indicates proteins not detected (ND). Six extractants, highlighted in red, were selected for further quantitative analysis. Selected reagents present in extraction solutions are labeled with colored dots.

Article Snippet: Anti-CDH3 (MAB861, R&D systems 1:500 in 10% (v/v) FBS in PBS) and Anti-CDH1 primary antibodies (AF648 R&D systems 1:200 in 10% (v/v) FBS in PBS) were added to the cells and incubated for 1 h. The cells were washed 3 times with 100 μl of 10% (v/v) FBS in PBS and incubated with secondary antibody conjugated to fluorophore (Goat anti-Mouse IgG (H; + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 or Donkey anti-Goat IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 at 1ug/ml in 10% (v/v) FBS in PBS).

Techniques: Protein-Protein interactions, Extraction, Staining, SDS Page, Transformation Assay, Quantitative Proteomics, Labeling

Journal: Molecular & Cellular Proteomics : MCP

Article Title: O -Mannose Glycosylations Influence E-Cadherin Functional Interactions

doi: 10.1016/j.mcpro.2026.101559

Figure Lengend Snippet: I-DIRT screen for CDH1. A , Depiction of the approach : BG1 WT cells and BG1 CDH1::HA cells were cultured in both light and heavy isotope-labeling media for label-swapped I-DIRT experiments. The resulting cell powders were combined in 1:1 (w:w) ratios for IP-MS analyses. Specific CDH1 interactors are enriched in one isotope-labeled channel in MS, while non-specific interactors are quantified comparably in both the heavy and light channels. BG1 CDH1::HA cells cultured in heavy-isotope media were designated ‘I-DIRT,’ while BG1 CDH1::HA cells cultured in light-isotope media were designated ‘I-DIRT swap.’ B , Specific interactors identified across six I-DIRT experimental conditions : The interactors are grouped based on how many times they were identified as specific interactors in six extractants. Orange lines represent interactions identified in this study, while gray lines indicate interactions retrieved from the STRING database . C , overlap of the I-DIRT interactor list with two previously published datasets ( , ): the panel on the right lists the 27 common interactors. Proteins are colored by their identification frequency, as in ( B ).

Article Snippet: Anti-CDH3 (MAB861, R&D systems 1:500 in 10% (v/v) FBS in PBS) and Anti-CDH1 primary antibodies (AF648 R&D systems 1:200 in 10% (v/v) FBS in PBS) were added to the cells and incubated for 1 h. The cells were washed 3 times with 100 μl of 10% (v/v) FBS in PBS and incubated with secondary antibody conjugated to fluorophore (Goat anti-Mouse IgG (H; + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 or Donkey anti-Goat IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 at 1ug/ml in 10% (v/v) FBS in PBS).

Techniques: Cell Culture, Quantitative Proteomics, Protein-Protein interactions, Labeling

Journal: Molecular & Cellular Proteomics : MCP

Article Title: O -Mannose Glycosylations Influence E-Cadherin Functional Interactions

doi: 10.1016/j.mcpro.2026.101559

Figure Lengend Snippet: Bioinformatic analyses of CDH1 interactors. Gene Ontologies (GO) ( A – C ) and Reactome pathways ( D ) enriched among the specific CDH1 interactors. A , enrichment of GO Cellular Components (CC) associated with CDH1 interactors. B , enrichment of GO Biological Processes (BP) associated with CDH1 interactors. C , enrichment of GO Molecular Functions (MF) associated with CDH1 interactors. D , the enriched Reactome pathways is shown on the left . The specific interactors involved in each pathway are detailed on the right . E , Localizations of select CDH1 interactors : proteins with annotated localizations at the cell surface or extracellular matrix are listed. The X-axis of panels ( A – C ) represents the proportion of proteins enriched in each GO pathway.

Article Snippet: Anti-CDH3 (MAB861, R&D systems 1:500 in 10% (v/v) FBS in PBS) and Anti-CDH1 primary antibodies (AF648 R&D systems 1:200 in 10% (v/v) FBS in PBS) were added to the cells and incubated for 1 h. The cells were washed 3 times with 100 μl of 10% (v/v) FBS in PBS and incubated with secondary antibody conjugated to fluorophore (Goat anti-Mouse IgG (H; + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 or Donkey anti-Goat IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 at 1ug/ml in 10% (v/v) FBS in PBS).

Techniques:

Journal: Molecular & Cellular Proteomics : MCP

Article Title: O -Mannose Glycosylations Influence E-Cadherin Functional Interactions

doi: 10.1016/j.mcpro.2026.101559

Figure Lengend Snippet: O -Man-dependent CDH1 interactome. A , Average log 2 fold change values for CDH1 interactors in TMTC deficient cell lines: colors represent the average log 2 FC values across the six IP conditions, with red indicating increased interaction with CDH1 and blue indicating decreased interaction with CDH1. The circle size indicates the number of IP conditions where the interactor was significantly changed in different TMTC KO cell lines (log 2 FC ≥ 1 or ≤ −1, and p-adj. value ≤ 0.05). B , Selected interactors across different co-enrichment groups, for distinct IP conditions. Symbols indicate statistical significance, with “∗” representing log 2 FC ≥ 1 or ≤ −1 and p-adj. value ≤ 0.05. C , Interactions dependent on O-mannosylation position :: ( upper - panel ) example interactions affected by O -Man on EC domain G-strands; ( middle - panel ) example interactions affected by O -Man on EC domain B- and G-strands together; ( lower - panel ) ANXA1 exhibits increased co-enrichment when O -Man is depleted from EC domain B-strands (see conditions 16 and 20, KO: TMTC2 ). D , cell adhesion ability in cell lines expressing CDH1 with varying O- Man modification statuses. Data are presented as mean ± SEM ( n = 6). Statistical significance is denoted as follows: ∗∗∗ p ≤ 0.001; ∗∗∗∗ p ≤ 0.0001.

Article Snippet: Anti-CDH3 (MAB861, R&D systems 1:500 in 10% (v/v) FBS in PBS) and Anti-CDH1 primary antibodies (AF648 R&D systems 1:200 in 10% (v/v) FBS in PBS) were added to the cells and incubated for 1 h. The cells were washed 3 times with 100 μl of 10% (v/v) FBS in PBS and incubated with secondary antibody conjugated to fluorophore (Goat anti-Mouse IgG (H; + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 or Donkey anti-Goat IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 at 1ug/ml in 10% (v/v) FBS in PBS).

Techniques: Expressing, Modification

Journal: Molecular & Cellular Proteomics : MCP

Article Title: O -Mannose Glycosylations Influence E-Cadherin Functional Interactions

doi: 10.1016/j.mcpro.2026.101559

Figure Lengend Snippet: Effects of TMTC knock - out on CDH1 and CDH3 abundance and localization. A , Western blot analysis of endogenous CDH3 abundance in BG1 cells with different TMTC KO statuses. B , Flow cytometry analysis of cell surface CDH1 and CDH3 : ( left ) representative histograms comparing fluorescence intensities in BG1 CDH1::HA cells ( green ), BG1 CDH1::HA/KO:TMTC1-4 cells ( pink ), and BG1 KO:CDH1 negative control cells ( grey ); signals normalized to mode; ( right ) Quantification of fold-change in median fluorescence intensity for surface CDH1 and CDH3 in BG1 CDH1::HA cells relative to BG1 CDH1::HA/KO:TMTC1-4 cells (n = 3). C , representative immunofluorescence images showing cellular localization of CDH1 ( green ) and CDH3 ( red ) in control BG1 CDH1::HA cells ( top panels ) and BG1 CDH1::HA/ KO :TMTC1-4 cells ( bottom panels ). Nuclei were counter-stained with DAPI ( blue ). Scale bar = 10 μm. D , Schematic model of the O-Man-dependent CDH1 interactome : some CDH1 interactors are modulated by O- Man, leading to their decreased or increased co-enrichment, based on changes e.g., in their affinity, localization, and/or abundance.

Article Snippet: Anti-CDH3 (MAB861, R&D systems 1:500 in 10% (v/v) FBS in PBS) and Anti-CDH1 primary antibodies (AF648 R&D systems 1:200 in 10% (v/v) FBS in PBS) were added to the cells and incubated for 1 h. The cells were washed 3 times with 100 μl of 10% (v/v) FBS in PBS and incubated with secondary antibody conjugated to fluorophore (Goat anti-Mouse IgG (H; + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 or Donkey anti-Goat IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 at 1ug/ml in 10% (v/v) FBS in PBS).

Techniques: Knock-Out, Western Blot, Flow Cytometry, Fluorescence, Negative Control, Immunofluorescence, Control, Staining

Journal: bioRxiv

Article Title: Bacterial internalins exploit E-cadherin to promote head and neck tumor metastasis and drug resistance

doi: 10.64898/2026.04.20.719623

Figure Lengend Snippet: (A-B) Co-immunoprecipitation (co-IP) assays. TIGK cell lysates were incubated with His-tagged recombinant InlA (rInlA) or InlB (rInlB; 20 μg/ml) for 3 hours at 4 °C, followed by incubation with Ni-NTA resin overnight. After washing (PBS, 0.05% Tween 20), bound proteins were eluted in Laemmli buffer and analyzed by immunoblotting with anti-His and anti-E-cadherin antibodies. (C) Co-IP assays using rInlB and recombinant Fc-E-cadherin (rE-cad). (D) Co-localization of InlA/InlB with E-cadherin. TIGK cells were incubated with FITC-labeled InlA or InlB (20 μg/ml, 2 h), followed by immunostaining with anti-E-cadherin and Alexa Fluor 594-conjugated secondary antibodies. Nuclei were counterstained with DAPI. Images were acquired using a Zeiss fluorescence microscope with a 63× oil-immersion objective. Scale bars: 20 μm. (E) E-cadherin knockdown efficiency in siRNA-transfected TIGK cells was confirmed by immunoblotting. (F) Quantification of FITC-InlB-positive cells was performed using ImageJ. (G) Binding of FITC-InlB to TIGK cells following E-cadherin knockdown was assessed by immuno-fluorescence microscopy. Scale bars: 20 μm. (H) Binding and internalization of FITC-labeled InlA or InlB in RKO and MOC1 cells were analyzed by immunofluorescence staining. Scale bars: 20 μm.

Article Snippet: Primary antibodies used in this study include anti-His (Invitrogen, cat. no. MA1-21315); E-cadherin (R&D Systems, cat. no. AF648); β-catenin (R&D Systems, cat. no. MAB1329); AKT (Cell Signaling Technology, cat. no. 9272); phospho-AKT (Ser473; cat. no. 9271); p38 (cat. no. 9212); phospho-p38 (Thr180/Tyr182; cat. no. 9211); JNK (cat. no. 9252); phospho-JNK (Thr183/Tyr185; cat. no. 9251); NF-κB p65 (cat. no. 8242); phospho-NF-κB p65 (Ser536; cat. no. 3033); c-Jun (cat. no. 9165); phospho-c-Jun (Ser63; cat. no. 9164); ERK1/2 (cat. no. 9102); phospho-ERK1/2 (Thr202/Tyr204; cat. no. 4370); and β-actin (cat. no. 3700) (all from Cell Signaling Technology unless otherwise indicated).

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Incubation, Recombinant, Western Blot, Labeling, Immunostaining, Fluorescence, Microscopy, Knockdown, Transfection, Binding Assay, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Bacterial internalins exploit E-cadherin to promote head and neck tumor metastasis and drug resistance

doi: 10.64898/2026.04.20.719623

Figure Lengend Snippet: (A) Schematic of E-cadherin showing five extracellular cadherin repeats (Ec1-5), transmembrane domain (TM), and cytoplasmic domain (CD). (B) SDS-PAGE of purified GST-tagged E-cadherin constructs: Ec1-5, Ec1, Ec1* (Ec1 P16E ), and Ec2-5. (C, E, G, I) SPR sensorgrams of InlB binding to Ec1-5 (C), Ec1 (E), Ec2-5 (G), and Ec1P16E (I) at multiple concentrations, showing association and dissociation kinetics. (D, F, H, J) Dose-dependent saturation binding of InlB to Ec1-5 (D), Ec1 (F), Ec2-5 (H), and Ec1 P16E (J). The equilibrium dissociation constant (K D ) was calculated for each binding experiment. (K) Structural modeling shows that Ec1 (cyan) interacts in the internal grove of InlB (tan, F5HDB6). (L) Close-up of the InlB-Ec1 interface highlights key residues and hydrogen bonds/salt bridges. Models of protein complexes between human E-cadherin Ec1 domain and InlA/InlB were generated using AlphaFold3. Structures were visualized, analyzed and figures were prepared using PyMol.

Article Snippet: Primary antibodies used in this study include anti-His (Invitrogen, cat. no. MA1-21315); E-cadherin (R&D Systems, cat. no. AF648); β-catenin (R&D Systems, cat. no. MAB1329); AKT (Cell Signaling Technology, cat. no. 9272); phospho-AKT (Ser473; cat. no. 9271); p38 (cat. no. 9212); phospho-p38 (Thr180/Tyr182; cat. no. 9211); JNK (cat. no. 9252); phospho-JNK (Thr183/Tyr185; cat. no. 9251); NF-κB p65 (cat. no. 8242); phospho-NF-κB p65 (Ser536; cat. no. 3033); c-Jun (cat. no. 9165); phospho-c-Jun (Ser63; cat. no. 9164); ERK1/2 (cat. no. 9102); phospho-ERK1/2 (Thr202/Tyr204; cat. no. 4370); and β-actin (cat. no. 3700) (all from Cell Signaling Technology unless otherwise indicated).

Techniques: SDS Page, Purification, Construct, Binding Assay, Generated

Journal: bioRxiv

Article Title: Bacterial internalins exploit E-cadherin to promote head and neck tumor metastasis and drug resistance

doi: 10.64898/2026.04.20.719623

Figure Lengend Snippet: (A-B) Wound-healing assay assessing HN30 cell migration following InlB treatment (30 μg/ml); representative images at 0 and 24 h (A) and ImageJ quantification of wound area relative to 0 h (B). Ctrl: PBS. (C-D) Transwell assays evaluating the effects of InlA or InlB on HN30 cell invasion after overnight treatment; invaded and migrated cells per field were quantified. (E-F) Wound-healing assays assessing HN30 cell migration following InlB or InlA treatment; wound area quantified using ImageJ. (G-H) qRT-PCR analysis of ZEB1 and MMP9 expression in HN30 cells treated with InlB or InlA for 24 h. (I) Immunoblot analysis of ZEB1 and MMP9 protein levels in HN30 cells following InlB or InlA treatment (24 h). (J) Immunoblot analysis of ZEB1 and MMP9 in HN3 cells transfected with E-cadherin siRNA and treated with or without InlB or InlA. (K-L) qRT-PCR analysis of ZEB1 and MMP9 expression in TIGK and HSC3 cells transfected with E-cadherin siRNA and treated with InlB.

Article Snippet: Primary antibodies used in this study include anti-His (Invitrogen, cat. no. MA1-21315); E-cadherin (R&D Systems, cat. no. AF648); β-catenin (R&D Systems, cat. no. MAB1329); AKT (Cell Signaling Technology, cat. no. 9272); phospho-AKT (Ser473; cat. no. 9271); p38 (cat. no. 9212); phospho-p38 (Thr180/Tyr182; cat. no. 9211); JNK (cat. no. 9252); phospho-JNK (Thr183/Tyr185; cat. no. 9251); NF-κB p65 (cat. no. 8242); phospho-NF-κB p65 (Ser536; cat. no. 3033); c-Jun (cat. no. 9165); phospho-c-Jun (Ser63; cat. no. 9164); ERK1/2 (cat. no. 9102); phospho-ERK1/2 (Thr202/Tyr204; cat. no. 4370); and β-actin (cat. no. 3700) (all from Cell Signaling Technology unless otherwise indicated).

Techniques: Wound Healing Assay, Migration, Quantitative RT-PCR, Expressing, Western Blot, Transfection

Journal: bioRxiv

Article Title: Bacterial internalins exploit E-cadherin to promote head and neck tumor metastasis and drug resistance

doi: 10.64898/2026.04.20.719623

Figure Lengend Snippet: InlA and InlB bind E-cadherin, triggering β-catenin stabilization and nuclear translocation, which induces EMT transcription factors (Slug, Twist, ZEB1) and MMP9, promoting metastasis. Concurrently, internalin-E-cadherin engagement activates downstream ROCK, p38 and JNK MAPK signaling, attenuating cisplatin-induced apoptosis through upregulation of Bcl-2 and suppression of Bax, ultimately conferring chemoresistance. These coordinated pathways mechanistically link P. gingivalis infection to cancer progression and therapeutic resistance. Green arrows denote activation; red blocked arrows denote inhibition.

Article Snippet: Primary antibodies used in this study include anti-His (Invitrogen, cat. no. MA1-21315); E-cadherin (R&D Systems, cat. no. AF648); β-catenin (R&D Systems, cat. no. MAB1329); AKT (Cell Signaling Technology, cat. no. 9272); phospho-AKT (Ser473; cat. no. 9271); p38 (cat. no. 9212); phospho-p38 (Thr180/Tyr182; cat. no. 9211); JNK (cat. no. 9252); phospho-JNK (Thr183/Tyr185; cat. no. 9251); NF-κB p65 (cat. no. 8242); phospho-NF-κB p65 (Ser536; cat. no. 3033); c-Jun (cat. no. 9165); phospho-c-Jun (Ser63; cat. no. 9164); ERK1/2 (cat. no. 9102); phospho-ERK1/2 (Thr202/Tyr204; cat. no. 4370); and β-actin (cat. no. 3700) (all from Cell Signaling Technology unless otherwise indicated).

Techniques: Translocation Assay, Infection, Activation Assay, Inhibition

Journal: bioRxiv

Article Title: Force transmission balance through adhesions determines multicellular handedness

doi: 10.64898/2026.04.04.716462

Figure Lengend Snippet: Inhibition of adherens junction reverses collective rotation. a , Schematic of our hypothesis that the balance of cell-cell adhesions and cell-substrate adhesions dictates the direction of collective rotation. When the relative contribution of cell-cell adhesions (e.g., adherens junctions, orange) is greater than that of cell-substrate adhesions (e.g., focal adhesions, blue), cells collectively rotate clockwise by kicking each other (a). When the relative contribution of cell-substrate adhesions is greater, cells collectively rotate counterclockwise by kicking the colony periphery (a’). b-c , Representative time courses of paired Caco-2 cells treated with normal IgG (b, control) and SHE78-7 (c). Time-lapse images of DIC (gray) and Lifeact (magenta) are shown. Cyan and red arrows are the vectors used to measure nuclear rotation and collective rotation, respectively. Scale bars: 20 μ m. d-e , Quantification of collective rotation (d) and relative nuclear rotation (e). Left panels: Time courses of cumulative rotation over 12 h. Black and gray lines indicate cumulative rotation angles in normal IgG treated and SHE78-7 treated cells, respectively. Error bars indicate standard errors. Right panels: Box plots of angular velocities calculated for 12 h of observation. Circles indicate angular velocities of individual cells. Crosses indicate average values. Sample sizes are shown in parentheses. **: p < 0.01; ***: p < 0.001 by t -test.

Article Snippet: The following reagents were used: 3 μM blebbistatin (SIGMA, #B0560-1MG); 50 μM nocodazole (SIGMA, #M1404); 1 μg/mL SHE78-7 (TAKARA, #M126); 1 μg/mL mouse normal IgG (SantaCruz, #sc-2025); 0.2% DMSO (negative control).

Techniques: Inhibition, Control