Journal: Cancers

Article Title: Pancreatic Cancers with High Grade Tumor Budding Exhibit Hallmarks of Diminished Anti-Tumor Immunity

doi: 10.3390/cancers13051090

Figure Lengend Snippet: Intratumoral (IT) immune profiling. Barplots displaying the IT leukocyte density in High Grade ( n = 54) versus Low Grade ( n = 57) tumor budding cases. The following immune cell markers were utilized (CD3, CD4, CD8, FOXP3, CD20, CD68, iNOS, CD163, DC-LAMP). Data are depicted as mean + standard error (SE). Differences between groups were analyzed using the Mann-Whitney U test. Only significant p -values (<0.05) are shown on the graph.

Article Snippet: They were double stained immunohistochemically for pancytokeratin (1:400, cytokeratin LMW, clone AE1/AE3, M3515, Dako-Agilent, Santa Clara, CA, USA) and each of the following: CD3 (1:400, clone SP7, ab16669, Abcam, Cambridge, UK), CD4 (1:100, clone CD4/4B12, M7310, Dako), CD8 (1:100, clone C8/144B, M7103, Dako), CD20 (1:100, clone L26, M0755, Dako), CD68 (1:100, clone KP1, M0814, Dako), DC-LAMP (1:100, CD208/DC-LAMP PA, 10527-H08H, Sino Biological, Beijing, China), iNOS (1:100, PAb, PA3-030A, Thermo Fisher Scientific, Waltham, MA, USA), CD163 (1:100, clone 10D6, NCL-CD163, Leica Biosystems AG, Muttenz, Switzerland) and FOXP3 (1:100, clone 236A/E7, ab20034, Abcam).

Techniques: MANN-WHITNEY

Journal: Cancers

Article Title: Pancreatic Cancers with High Grade Tumor Budding Exhibit Hallmarks of Diminished Anti-Tumor Immunity

doi: 10.3390/cancers13051090

Figure Lengend Snippet: Stromal (S) immune profiling. Barplots displaying the S leukocyte density in High Grade ( n = 54) versus Low Grade ( n = 57) tumor budding cases. The following immune cell markers were utilized (CD3, CD4, CD8, FOXP3, CD20, CD68, iNOS, CD163, DC-LAMP). Data are depicted as mean + SE. Differences between groups were analyzed using the Mann-Whitney U test. Only significant P values are shown on the graph.

Article Snippet: They were double stained immunohistochemically for pancytokeratin (1:400, cytokeratin LMW, clone AE1/AE3, M3515, Dako-Agilent, Santa Clara, CA, USA) and each of the following: CD3 (1:400, clone SP7, ab16669, Abcam, Cambridge, UK), CD4 (1:100, clone CD4/4B12, M7310, Dako), CD8 (1:100, clone C8/144B, M7103, Dako), CD20 (1:100, clone L26, M0755, Dako), CD68 (1:100, clone KP1, M0814, Dako), DC-LAMP (1:100, CD208/DC-LAMP PA, 10527-H08H, Sino Biological, Beijing, China), iNOS (1:100, PAb, PA3-030A, Thermo Fisher Scientific, Waltham, MA, USA), CD163 (1:100, clone 10D6, NCL-CD163, Leica Biosystems AG, Muttenz, Switzerland) and FOXP3 (1:100, clone 236A/E7, ab20034, Abcam).

Techniques: MANN-WHITNEY

Journal: Cancers

Article Title: Pancreatic Cancers with High Grade Tumor Budding Exhibit Hallmarks of Diminished Anti-Tumor Immunity

doi: 10.3390/cancers13051090

Figure Lengend Snippet: Multivariate Cox regression analysis with model performance is summarized.

Article Snippet: They were double stained immunohistochemically for pancytokeratin (1:400, cytokeratin LMW, clone AE1/AE3, M3515, Dako-Agilent, Santa Clara, CA, USA) and each of the following: CD3 (1:400, clone SP7, ab16669, Abcam, Cambridge, UK), CD4 (1:100, clone CD4/4B12, M7310, Dako), CD8 (1:100, clone C8/144B, M7103, Dako), CD20 (1:100, clone L26, M0755, Dako), CD68 (1:100, clone KP1, M0814, Dako), DC-LAMP (1:100, CD208/DC-LAMP PA, 10527-H08H, Sino Biological, Beijing, China), iNOS (1:100, PAb, PA3-030A, Thermo Fisher Scientific, Waltham, MA, USA), CD163 (1:100, clone 10D6, NCL-CD163, Leica Biosystems AG, Muttenz, Switzerland) and FOXP3 (1:100, clone 236A/E7, ab20034, Abcam).

Techniques:

Journal: The Journal of international medical research

Article Title: Expression of dendritic cell lysosome-associated membrane protein and dendritic cell-specific intercellular adhesion molecule-3 grabbing nonintegrin in condyloma acuminatum lesions.

doi: 10.1177/0300060513476991

Figure Lengend Snippet: Figure 1. Immunohistochemical staining of dendritic cell lysosome-associated membrane protein (DC-LAMP) in (A) normal foreskin tissue and (B) condyloma acuminatum lesion; arrows show DC-LAMP in epidermis and dermis. Immunohistochemical staining of dendritic cell-specific intercellular adhesion molecule- 3 grabbing nonintegrin (DC-SIGN) protein in (C) normal foreskin tissue and (D) condyloma acuminatum lesion; arrows show DC-SIGN in epidermis and dermis.

Article Snippet: Separated proteins were then transferred onto a polyvinylidene fluoride (PVDF) membrane at 40V for 2 h. After blocking with 5% low-fat dry milk for 1 h, the PVDF membranes were incubated with at Glasgow University Library on May 20, 2015imr.sagepub.comDownloaded from a monoclonal antihuman DC-LAMP antibody (1 : 500 dilution; R&D Systems), monoclonal antihuman DC-SIGN antibody (1 : 500 dilution; R&D Systems), or mouse antihuman b-actin antibody (C4) (1 : 400 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4 C. Membranes were washed with 50mM Tris,150mM NaCl, and 0.05% Tween 20, pH 7.6 (TBST) five times for 5min, then incubated with horseradish peroxidaselabelled goat antirabbit immunoglobulin (HþL) antibody (1 : 50 dilution; Beyotime) for 1 h at 37 C. Blots were washed three times for 5min in TBST and developed with DAB to visualize the protein bands.

Techniques: Immunohistochemical staining, Staining, Membrane

Journal: Scientific Reports

Article Title: LAMP3 transfer via extracellular particles induces apoptosis in Sjögren’s disease

doi: 10.1038/s41598-023-28857-w

Figure Lengend Snippet: LAMP3-positive material is released in minor salivary glands from SjD patients. A representative immunofluorescent image of minor salivary glands from SjD patients ( n = 11) stained with anti-LAMP3 antibody (scale bar: 50 μm). The image shows accumulation of LAMP3 in the lumen of the gland (the arrows) and in the epithelial cells (the arrowheads).

Article Snippet: EPs collected from LAMP3-overexpressing and control A253 cells were incubated at 4 °C overnight with exosome isolation magnetic beads (Thermo Fisher Scientific), which were conjugated with biotin anti-LAMP3 monoclonal antibody (clone:104G4; Novus Biological) according to the manufacturer’s instructions.

Techniques: Staining

Journal: Scientific Reports

Article Title: LAMP3 transfer via extracellular particles induces apoptosis in Sjögren’s disease

doi: 10.1038/s41598-023-28857-w

Figure Lengend Snippet: Culture medium of LAMP3-overexpressing A253 cells induce apoptosis in naïve A253 cells. A253 cells were transfected with control empty plasmid, LAMP3 -encoding plasmid or LAMP1 -encoding plasmid. ( A ) Representative Western blots of transfected A253 cells (uncropped images are provided in Supplementary Fig. ) and schematic methods of the following in vitro assays. ( B ) Naïve A253 cells were treated with culture medium of control or LAMP3-overexpressing A253 cells for 48 h ( n = 3). ( C ) Naïve A253 cells were treated with culture medium of control, LAMP1-overexpressing, or LAMP3-overexpressing A253 cells or with post-ultracentrifugation supernatant or post-ultracentrifugation pellet from LAMP3-overexpressing A253 cells for 48 h ( n = 4). Apoptotic cells in naïve cell culture were determined by flow cytometry using APC Annexin V/7-AAD. Graphs show difference in mean (± SD) number of Annexin V + /7-AAD + cells in cell culture compared with control. ** p < 0.01 (one-way ANOVA).

Article Snippet: EPs collected from LAMP3-overexpressing and control A253 cells were incubated at 4 °C overnight with exosome isolation magnetic beads (Thermo Fisher Scientific), which were conjugated with biotin anti-LAMP3 monoclonal antibody (clone:104G4; Novus Biological) according to the manufacturer’s instructions.

Techniques: Transfection, Control, Plasmid Preparation, Western Blot, In Vitro, Cell Culture, Flow Cytometry

Journal: Scientific Reports

Article Title: LAMP3 transfer via extracellular particles induces apoptosis in Sjögren’s disease

doi: 10.1038/s41598-023-28857-w

Figure Lengend Snippet: A fraction of extracellular particles from LAMP3-overexpressing A253 cells induces apoptosis in naïve A253 cells. ( A ) Schematic methods. Two fractions of extracellular particles (EPs) were separated from culture medium of control, LAMP3-overexpressing, or LAMP1-overexpressing A253 cells using centrifugation in combination with or without total exosome isolation reagent. Naïve A253 cells were treated with ( B ) EP fraction 2, ( C ) EP fraction 1 or ( D ) heat-denatured (heat-treated) EP fraction 1. Apoptotic cells in naïve cell culture were determined by flow cytometry using APC Annexin V/7-AAD 72 h after incubation. Graphs show difference in mean (± SD) number of Annexin V + /7-AAD + cells in cell culture compared with control ( n = 3 for all the experiments). ** p < 0.01 (one-way ANOVA).

Article Snippet: EPs collected from LAMP3-overexpressing and control A253 cells were incubated at 4 °C overnight with exosome isolation magnetic beads (Thermo Fisher Scientific), which were conjugated with biotin anti-LAMP3 monoclonal antibody (clone:104G4; Novus Biological) according to the manufacturer’s instructions.

Techniques: Control, Centrifugation, Isolation, Cell Culture, Flow Cytometry, Incubation

Journal: Scientific Reports

Article Title: LAMP3 transfer via extracellular particles induces apoptosis in Sjögren’s disease

doi: 10.1038/s41598-023-28857-w

Figure Lengend Snippet: Extracellular particles from LAMP3-overexpressing cells contain LAMP3. ( A ) Western blot analysis of EPs separated from the culture medium of control, LAMP3-overexpressing, and LAMP1-overexpressing A253 cells using centrifugation in combination with or without total exosome isolation reagent. Equal volume of protein loaded in each lane. Lower panel shows same membrane as the one used in Western blot analysis but stained with Reversible Protein Stain Kit. Uncropped images are provided in Supplementary Fig. . ( B ) Cell surface LAMP3 expression was analyzed by flow cytometry (bold line: LAMP3 staining, filled area with dashed line: isotype control) and under an immunofluorescent microscopy (scale bar: 100 μm) in control, LAMP1-overexpressing, and LAMP3-overexpressing A253 cells 48 h post-transfection. ( C ) EPs derived from control and LAMP3-overexpressing A253 cells were immunoprecipitated using anti-LAMP3 monoclonal antibody-conjugated beads. Presence of LAMP3 protein on EP membrane surface was determined by analyzing beads stained with anti-LAMP3 polyclonal antibody using flow cytometry. Graph showing difference in mean (± SD) percentage of LAMP3-positive beads compared with beads bound to isotype control ( n = 4). ** p < 0.01 (one-way ANOVA).

Article Snippet: EPs collected from LAMP3-overexpressing and control A253 cells were incubated at 4 °C overnight with exosome isolation magnetic beads (Thermo Fisher Scientific), which were conjugated with biotin anti-LAMP3 monoclonal antibody (clone:104G4; Novus Biological) according to the manufacturer’s instructions.

Techniques: Western Blot, Control, Centrifugation, Isolation, Membrane, Staining, Expressing, Flow Cytometry, Microscopy, Transfection, Derivative Assay, Immunoprecipitation

Journal: Scientific Reports

Article Title: LAMP3 transfer via extracellular particles induces apoptosis in Sjögren’s disease

doi: 10.1038/s41598-023-28857-w

Figure Lengend Snippet: Extracellular particles from LAMP3-overexpressing transfer LAMP3 to naïve cells. ( A ) Western blot analysis of naïve A253 cells treated with EPs from control or LAMP3-overexpressing A253 cells. “※” indicates non-specific bands. ( B ) Western blot analysis of LAMP3-GFP expression in A253 cells transfected with empty plasmid (control) or pLenti-LAMP3-mGFP plasmid (A253-LAMP3-mGFP cells). ( C ) Western blot analysis of EPs derived from A253-LAMP3-mGFP cells. ( D ) A253-LAMP3-mGFP cells were imaged in real time to visualize EP release. A particle containing LAMP3-mGFP is seen being released from the cell into the extracellular environment (scale bar: 5 μm). ( E ) Naïve A253 cells were treated with LAMP3-mGFP–containing particle and imaged in real time to visualize particle uptake. Uptake of a LAMP3-mGFP–containing particle by a naïve cell and subsequent membrane blebbing are shown (scale bar: 10 μm). Uncropped images of Western blots are provided in Supplementary Fig. .

Article Snippet: EPs collected from LAMP3-overexpressing and control A253 cells were incubated at 4 °C overnight with exosome isolation magnetic beads (Thermo Fisher Scientific), which were conjugated with biotin anti-LAMP3 monoclonal antibody (clone:104G4; Novus Biological) according to the manufacturer’s instructions.

Techniques: Western Blot, Control, Expressing, Transfection, Plasmid Preparation, Derivative Assay, Membrane

Journal: Scientific Reports

Article Title: LAMP3 transfer via extracellular particles induces apoptosis in Sjögren’s disease

doi: 10.1038/s41598-023-28857-w

Figure Lengend Snippet: Transfection of recombinant LAMP3 protein induces apoptosis in A253 cells. ( A ) Representative Western blots of A253 cells transfected with recombinant LAMP3 (rLAMP3), recombinant LAMP1 (rLAMP1), or control FLAG peptide (uncropped images are provided in Supplementary Fig. ). ( B ) Apoptotic cells were determined in A253 cell culture by flow cytometry using APC Annexin V/7-AAD 14 h after transfection. Graph showing difference in mean (± SD) number of Annexin V + /7-AAD + cells in A253 cell culture transfected with rLAMP3 or rLAMP1 compared with control. ( C ) A253 cells were transfected with rLAMP3, heat-treated rLAMP3, or FLAG peptide. Apoptotic cells were determined in A253 cell culture by flow cytometry using APC Annexin V/7-AAD 14 h after transfection. Graph showing difference in mean (± SD) number of Annexin V + /7-AAD + cells in A253 cell culture transfected with rLAMP3 or heat-treated rLAMP3 compared with control. ( D ) A253 cells were treated with control FLAG peptide or rLAMP3 alone (without transfection reagent) or transfected with control FLAG peptide or rLAMP3 with transection reagent. After 14 h, number of apoptotic cells was determined by flow cytometry using APC Annexin V/7-AAD. Graph showing difference in mean (± SD) number of Annexin V + /7-AAD + cells in cell culture treated with rLAMP3 alone or transfected with FLAG peptide or rLAMP3 compared with FLAG peptide alone. ** p < 0.01 (one-way ANOVA, n = 4 for all the experiments).

Article Snippet: EPs collected from LAMP3-overexpressing and control A253 cells were incubated at 4 °C overnight with exosome isolation magnetic beads (Thermo Fisher Scientific), which were conjugated with biotin anti-LAMP3 monoclonal antibody (clone:104G4; Novus Biological) according to the manufacturer’s instructions.

Techniques: Transfection, Recombinant, Western Blot, Control, Cell Culture, Flow Cytometry

Journal: Scientific Reports

Article Title: LAMP3 transfer via extracellular particles induces apoptosis in Sjögren’s disease

doi: 10.1038/s41598-023-28857-w

Figure Lengend Snippet: LAMP3 transfer via extracellular particles induces caspase-dependent apoptosis. ( A ) Naïve A253 cells were treated with EPs from control and LAMP3-overexpressing A253 cells in combination with or without zVAD-fmk (zVAD), a pan-caspase inhibitor. Apoptotic cells were determined by flow cytometry using APC Annexin V/7-AAD 72 h after incubation. Graph showing difference in mean (± SD) number of Annexin V + /7-AAD + cells in naïve A253 cell culture compared with control ( n = 5). ( B ) Naïve A253 cells were treated with EPs from control and LAMP3-overexpressing A253 cells for 6 h. Galectin-3 puncta formation in treated A253 cell culture was visualized by immunofluorescence (scale bar: 50 μm). Graph showing percentage of galectin-3 puncta–positive cells per 100 treated A253 cells ( n = 3). ** p < 0.01 (one-way ANOVA).

Article Snippet: EPs collected from LAMP3-overexpressing and control A253 cells were incubated at 4 °C overnight with exosome isolation magnetic beads (Thermo Fisher Scientific), which were conjugated with biotin anti-LAMP3 monoclonal antibody (clone:104G4; Novus Biological) according to the manufacturer’s instructions.

Techniques: Control, Flow Cytometry, Incubation, Cell Culture, Immunofluorescence

Journal: Scientific Reports

Article Title: LAMP3 transfer via extracellular particles induces apoptosis in Sjögren’s disease

doi: 10.1038/s41598-023-28857-w

Figure Lengend Snippet: Graphical summary. Extracellular particles containing LAMP3 are released by LAMP3-overexpressing cells and taken up by a neighboring LAMP3-naïve cell. This results in LAMP3 protein transfer to the cytoplasm of the naïve cell. Here, LAMP3 induces lysosomal membrane permeabilization (LMP), leading to apoptotic cell death.

Article Snippet: EPs collected from LAMP3-overexpressing and control A253 cells were incubated at 4 °C overnight with exosome isolation magnetic beads (Thermo Fisher Scientific), which were conjugated with biotin anti-LAMP3 monoclonal antibody (clone:104G4; Novus Biological) according to the manufacturer’s instructions.

Techniques: Membrane