Journal: Journal of proteome research

Article Title: Rapid Characterization of Candidate Biomarkers for Pancreatic Cancer Using Cell Microarrays (CMAs)

doi: 10.1021/pr300483r

Figure Lengend Snippet: Immunocytochemical staining of TMPRSS4. In the top left panel, the domain structure of TMPRSS4 is shown, where TM refers to the transmembrane domain. The left bottom panel shows a CMA slide stained with a monoclonal antibody directed against TMPRSS4. The panel on the right shows a magnified view of four cell lines showing different levels of expression of TMPRSS4.

Article Snippet: CMA cell microarray Ep-CAM epithelial cell adhesion molecule CA19-9 carbohydrate antigen 19-9 HLA human leukocyte antigen TMPRSS4 transmembrane protease, serine 4 IHC immunohistochemistry ICC immunocytochemistry TMA tissue microarray ATCC American Type Culture Collection

Techniques: Staining, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Interleukin-17A Promotes Aortic Endothelial Cell Activation via Transcriptionally and Post-translationally Activating p38 Mitogen-activated Protein Kinase (MAPK) Pathway *

doi: 10.1074/jbc.M115.690081

Figure Lengend Snippet: Hyperlipidemia stimuli induce IL-17 and its receptor subunit expression. A, the plasma IL-17 concentration was determined by ELISA. WT and ApoE−/− mice were fed with normal chow or high fat diet (HFD) for 8 weeks starting at 8 weeks of age. Plasma from mice was collected for ELISA analysis. B, IL-17RA mRNA expression in MAECs was determined by qRT-PCR. MAECs were treated with ox-LDL (50 μg/ml) for 24 h, and the IL-17RA mRNA expression was quantified. IL-17RA expression was significantly increased with ox-LDL treatment relative to the control cells. Data were normalized to β-actin. C and D, the protein expressions of IL-17RA and IL-17RC in HAECs were quantified by flow cytometry. HAECs were treated with LDL (50 μg/ml) or ox-LDL (50 μg/ml) for 24 h. C, IL-17RA expression was increased with ox-LDL treatment in HAECs. D, ox-LDL treatment significantly increased IL-17RC expression in HAECs. Data are presented as means ± S.E. (error bars) (n = 3). *, p < 0.05; **, p < 0.01 versus untreated control cells.

Article Snippet: HAECs (passage 9) were cultured in 24-well plates and treated with human IL-17 (100 ng/ml; R&D Systems).

Techniques: Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Flow Cytometry

Journal: The Journal of Biological Chemistry

Article Title: Interleukin-17A Promotes Aortic Endothelial Cell Activation via Transcriptionally and Post-translationally Activating p38 Mitogen-activated Protein Kinase (MAPK) Pathway *

doi: 10.1074/jbc.M115.690081

Figure Lengend Snippet: Treatment of HAECs with cytokine IL-17 increases the non-treated monocyte adhesion to HAECs. A, IL-17-treated HAECs have increased THP-1 cell adhesion. HAECs were treated with IL-17 (100 ng/ml) for the indicated time, and untreated labeled monocytes were allowed to adhere onto the endothelial monolayer. IL-17 treatment increased THP-1 monocytic cell adhesion onto HAECs in a time-dependent manner. The adhesion is shown as a percentage of the control group. B, untreated primary human monocyte adhesion to HAECs treated with IL-17 gradually increased up to 24 h. C, fluorescence microscopic images of labeled primary human monocyte adhered to HAECs. Data are presented as means ± S.E. (error bars) (n = 3). *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus control.

Article Snippet: HAECs (passage 9) were cultured in 24-well plates and treated with human IL-17 (100 ng/ml; R&D Systems).

Techniques: Labeling, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Interleukin-17A Promotes Aortic Endothelial Cell Activation via Transcriptionally and Post-translationally Activating p38 Mitogen-activated Protein Kinase (MAPK) Pathway *

doi: 10.1074/jbc.M115.690081

Figure Lengend Snippet: Depletion of IL-17 gene in ApoE−/− mice significantly decreases in vivo leukocyte rolling and adhesion to endothelium. ApoE−/− mice and ApoE−/−/IL-17A−/− mice were fed with a high fat diet for 3 weeks before intravital microscopy analysis was performed. A, intravital microscopy was used to determine the endothelial activation by determining leukocyte rolling and adhesion to endothelium in vivo. CCD, charge-coupled device. B, mean vessel diameters of venules were not significantly different between ApoE−/− and ApoE−/−/IL-17A−/− mice used for the intravital microscopy study. C, number of cells that rolled past an imaginary line that was perpendicular to the vessels during a 1-min period were not significant in both animal groups used in the study. D, the number of cells adhered to 100 μm of endothelium along the vessel for 1 min was significantly reduced in IL-17-null mice relative to the controls. Data are presented as means ± S.E. (error bars) (n = 7–8; five vessels per mouse were recorded). *, p < 0.05 (significant); n.s., not significant.

Article Snippet: HAECs (passage 9) were cultured in 24-well plates and treated with human IL-17 (100 ng/ml; R&D Systems).

Techniques: In Vivo, Intravital Microscopy, Activation Assay

Journal: The Journal of Biological Chemistry

Article Title: Interleukin-17A Promotes Aortic Endothelial Cell Activation via Transcriptionally and Post-translationally Activating p38 Mitogen-activated Protein Kinase (MAPK) Pathway *

doi: 10.1074/jbc.M115.690081

Figure Lengend Snippet: IL-17 induces the gene expression of four proinflammatory cytokines and chemokines, namely IL-6, GM-CSF, CXCL1, and CXCL2, in mouse and human aortic endothelial cells. The expressions of 84 genes related to EC biology were profiled by using the Mouse Endothelial Cell Biology RT2 Profiler PCR Array. MAECs were treated with IL-17 for 24 h and used in the Mouse Endothelial Cell Biology RT2 Profiler PCR Array. A, 84 genes assessed by the array. B, scatter plot of the changes of gene expression in IL-17-treated MAECs. Expressions of four genes were dramatically induced by IL-17 treatment; blue lines indicate a -fold change of 4. C, IL-17-mediated increases in the expressions of CXCL1, CXCL2, IL-6, and CSF2 (GM-CSF) were further confirmed in MAECs by quantitative PCR. Data are presented as means ± S.E. (n = 5). *, p < 0.05; **, p < 0.01; ***p < 0.001 versus control. D, human IL-17 treatment significantly enhanced the expressions of CXCL1, CXCL2, IL-6, and CSF2 in HAECs treated with IL-17 (100 ng/ml) for 12 h as detected by qRT-PCR. Data were normalized to β-actin expression and are presented as means ± S.E. (n = 3). *, p < 0.05 versus control. E, the blockage of proinflammatory cytokines IL-6 and GM-CSF and chemokines CXCL1 and CXCL2 with specific antibodies decreased non-treated monocyte adhesion to IL-17-stimulated endothelial cells. Endothelial cells were treated with IL-17, and the blocking antibodies against CXCL1/2, IL-6, and/or GM-CSF were added 30 min prior to the adhesion assay. Antibodies against CXCL1/2, IL-6, and GM-CSF reversed the effect of IL-17 on increasing monocyte adhesion to endothelial cells relative to the IgG control. F, fluorescence microscopic image of labeled non-treated THP-1 cells adhered to endothelial cells. Data are presented as means ± S.E. (error bars) (n = 3). *, p < 0.05 versus control; #, p < 0.05 versus IL-17-treated group.

Article Snippet: HAECs (passage 9) were cultured in 24-well plates and treated with human IL-17 (100 ng/ml; R&D Systems).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Blocking Assay, Cell Adhesion Assay, Fluorescence, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Interleukin-17A Promotes Aortic Endothelial Cell Activation via Transcriptionally and Post-translationally Activating p38 Mitogen-activated Protein Kinase (MAPK) Pathway *

doi: 10.1074/jbc.M115.690081

Figure Lengend Snippet: IL-17 and IL-6 synergistically induce ICAM-1 up-regulation in HAECs. HAECs were treated with IL-6 (20 ng/ml), IL-17 (50 ng/ml), or IL-6 (20 ng/ml) + IL-17 (50 ng/ml) for 6 h. After the treatment, cells were collected, and ICAM-1 expression was quantified by a flow cytometry method with fluorescence-conjugated anti-ICAM-1 antibody. A, histograms showing an increase of ICAM-1 expression after IL-17 (50 ng/ml) treatment that was further increased when the cells were treated with IL-6 (20 ng/ml) plus IL-17 (50 ng/ml). B, bar graph showing quantification of ICAM-1 expression in HAECs. Data are presented as mean ± S.E. (error bars) (n = 3). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: HAECs (passage 9) were cultured in 24-well plates and treated with human IL-17 (100 ng/ml; R&D Systems).

Techniques: Expressing, Flow Cytometry, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Interleukin-17A Promotes Aortic Endothelial Cell Activation via Transcriptionally and Post-translationally Activating p38 Mitogen-activated Protein Kinase (MAPK) Pathway *

doi: 10.1074/jbc.M115.690081

Figure Lengend Snippet: Mouse aortic microarray results indicate that IL-17 up-regulates the expressions of many genes in mouse aortas including IL-17RA, endothelial cell adhesion molecules, and MAPK pathway components. A, mouse aortic RNAs were isolated from ApoE−/−mice and IL-17−/−/ApoE−/− mice fed with a high fat diet for 3 weeks and subjected to Affymetrix microarray analysis. B, volcano plot (scatter plot) of -fold change versus p value for all gene transcripts determined by the Affymetrix GeneChip Mouse Gene 2.0ST Arrays in mouse aortas from ApoE−/−mice and IL-17−/−/ApoE−/− mice fed with a high fat diet for 3 weeks. The volcano plot shows that there are numerous genes that were modified with statistical significance in IL-17−/−/ApoE−/− mouse aortas relative to ApoE−/− mouse aortas. C, the heat map and -fold changes of IL-17 receptor signaling genes. D, heat map and -fold changes of endothelial adhesion molecule genes. E, heat map and -fold changes of p38 MAPK (MAPK14) pathway genes. The values with p < 0.05 are highlighted in red.

Article Snippet: HAECs (passage 9) were cultured in 24-well plates and treated with human IL-17 (100 ng/ml; R&D Systems).

Techniques: Microarray, Isolation, Modification

Journal: The Journal of Biological Chemistry

Article Title: Interleukin-17A Promotes Aortic Endothelial Cell Activation via Transcriptionally and Post-translationally Activating p38 Mitogen-activated Protein Kinase (MAPK) Pathway *

doi: 10.1074/jbc.M115.690081

Figure Lengend Snippet: Depletion of IL-17 in ApoE−/− mice decreases the phosphorylation of p38 MAPK but not the phosphorylation of JNK MAPK nor the phosphorylation of eNOS in mouse aortas, and IL-17 also induces the phosphorylation of p38 MAPK in vitro. A, mouse aortic proteins were isolated from ApoE−/−mice and IL-17−/−/ApoE−/− mice fed with a high fat diet for 3 weeks. Representative Western blotting analyses of phosphorylated and total p38, phosphorylated and total JNK, and phosphorylated and total eNOS, and β-actin expressions are shown. B, quantification of total p38. C, quantification of total JNK. D, quantification of the phosphorylated p38. E, quantification of phosphorylated JNK. F, schematic representation of how IL-17 transcriptionally and post-translationally activates the p38 MAPK pathway but not JNK MAPK pathway. G, quantitation of phosphorylated eNOS (Ser-1177). H, quantification of phosphorylated eNOS (Thr-495). I, IL-17 treatment induced the phosphorylation of p38 MAPK and its upstream kinase Map2k3 in HAECs. Representative Western blotting analyses of phosphorylated p38 MAPK, total p38 MAPK, phosphorylated Map2k3, and total Map2k3 are shown. J, IL-17 treatment did not induce JNK and its upstream kinase Map2k4 in HAECs. Western blotting analyses of phosphorylated JNK, total JNK, phosphorylated Map2k4, and total Map2k4 are represented. Data are presented as means ± S.E. (error bars). *, p < 0.05 (significant); n.s., not significant.

Article Snippet: HAECs (passage 9) were cultured in 24-well plates and treated with human IL-17 (100 ng/ml; R&D Systems).

Techniques: In Vitro, Isolation, Western Blot, Quantitation Assay

Journal: The Journal of Biological Chemistry

Article Title: Interleukin-17A Promotes Aortic Endothelial Cell Activation via Transcriptionally and Post-translationally Activating p38 Mitogen-activated Protein Kinase (MAPK) Pathway *

doi: 10.1074/jbc.M115.690081

Figure Lengend Snippet: Inhibition of IL-17-mediated phosphorylation of p38 MAPK attenuates the inflammation and endothelial activation in human aortic endothelial cells in vitro. A, p38 MAPK deficiency decreased IL-6 and ICAM-1 gene expressions. Microarray database mining analysis was carried out by analyzing the National Institutes of Health NCBI GEO data set GSE7342, which studied the role of p38α in regulating the gene expression in mouse embryonic development. B, inhibition of p38 MAPK decreased the expression of ICAM-1 in IL-17-treated HAECs. HAECs were pretreated for 1 h with p38 inhibitor (SB203580; 10 μm) followed by IL-17 (100 ng/ml) treatment for 6 h. The cells were stained with fluorescent dye allophycocyanin-conjugated ICAM-1 antibody for 30 min and analyzed by flow cytometry with IgG isotype control. C and D, the HAECs were pretreated with SB203580 (10 μm) for 1 h followed by IL-17 treatment (100 ng/ml) for 24 h. The CXCL1/2 gene expression was measured by quantitative PCR. C, p38 MAPK inhibitor significantly ameliorated IL-17-induced CXCL1 gene expression. D, p38 MAPK inhibitor significantly ameliorated CXCL2 gene expression mediated by IL-17. E, p38 MAPK inhibitor significantly attenuated the IL-17-induced non-treated monocyte adhesion to HAECs. The HAECs were pretreated with SB203580 (10 μm) for 1 h prior to incubation with IL-17 (100 ng/ml) for 24 h. Data are presented as means ± S.E. (error bars). ***, p < 0.001; **, p < 0.01; *, p < 0.05.

Article Snippet: HAECs (passage 9) were cultured in 24-well plates and treated with human IL-17 (100 ng/ml; R&D Systems).

Techniques: Inhibition, Activation Assay, In Vitro, Microarray, Expressing, Staining, Flow Cytometry, Real-time Polymerase Chain Reaction, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Interleukin-17A Promotes Aortic Endothelial Cell Activation via Transcriptionally and Post-translationally Activating p38 Mitogen-activated Protein Kinase (MAPK) Pathway *

doi: 10.1074/jbc.M115.690081

Figure Lengend Snippet: Deficiency of IL-17A in ApoE−/− mice modulates the lipid profile but does not significantly affect atherosclerotic lesion formation. A, mouse experimental design. Male ApoE−/− mice and IL-17−/−/ApoE−/− mice were put on either a high fat diet or regular chow diet at 8 weeks of age for 3 weeks. B, body weight, heart weight, and spleen weight of ApoE−/− mice and IL-17−/−/ApoE−/− mice that were on either a normal chow diet or a high fat diet for 3 weeks. Data are presented as means ± S.E. (n = 7–10). C, the plasma lipid profiles of mice that were fed a high fat (HF) diet or normal chow diet for 3 weeks. FFA, free fatty acids. Data are presented as means ± S.E. (n = 7–10). *, p < 0.05. D, atherosclerotic plaque formation in the whole aorta was assessed with Sudan IV staining. The upper panel represents the images of 3-week-diet mouse group aortas stained with Sudan IV. The lower panel indicates the quantification of atherosclerotic area percentage in the whole aorta of the 3-week-diet mouse group. Data are presented as means ± S.E. (n = 4–11). E, atherosclerotic plaque in aortic sinus was determined by Oil Red O staining of aortic cross-sections. The upper panel represents the images of aortic sinus cross-section stained with Oil Red O in 3-week-diet mouse groups. The lower panel shows the quantification of atherosclerotic area percentage in the aortic sinus of 3-week-diet mouse groups. Data are presented as means ± S.E. (error bars) (n = 5).

Article Snippet: HAECs (passage 9) were cultured in 24-well plates and treated with human IL-17 (100 ng/ml; R&D Systems).

Techniques: Staining

Journal: The Journal of Biological Chemistry

Article Title: Interleukin-17A Promotes Aortic Endothelial Cell Activation via Transcriptionally and Post-translationally Activating p38 Mitogen-activated Protein Kinase (MAPK) Pathway *

doi: 10.1074/jbc.M115.690081

Figure Lengend Snippet: The proposed novel working model. IL-17 promotes endothelial cell activation by inducing p38 MAPK, which leads to up-regulation of proinflammatory cytokines, chemokines, and endothelial cell adhesion molecules.

Article Snippet: HAECs (passage 9) were cultured in 24-well plates and treated with human IL-17 (100 ng/ml; R&D Systems).

Techniques: Activation Assay

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Integrated Systems Analysis of the Murine and Human Pancreatic Cancer Glycomes Reveals a Tumor-Promoting Role for ST6GAL1

doi: 10.1016/j.mcpro.2021.100160

Figure Lengend Snippet: Transcriptomic analysis identifies ST6GAL1 and ST3GAL3 as enriched in the cancerous ducts. A , biosynthetic pathways for glycans underlying select lectin signatures are shown. ST3GALs are responsible for transferring α-2,3-sialosides and ST6GAL1/2 for α-2,6-sialosides, and MGAT3 for bisecting GlcNAc. The glycans are annotated following the Symbolic Nomenclature for Glycans. B , transcriptomic analysis assessing the mRNA levels of select glycosyltransferases between normal adjacent and matched cancerous tissues within the same patient. C , uMAP plots representing the cells isolated from patients with PDAC (n = 24) and normal pancreata (n = 11) pooled on single-cell sequencing. The clusters representing the normal ductal cells and tumor ductal cells are highlighted. D , violin plot showing the comparison of ST6Gal1 levels in normal ( green ) versus PDAC ( blue ) ductal clusters. The clusters in each group are combined. The violin plots showing the comparison of ST6Gal1 and ST3GAL1 levels in normal ( green ) versus PDAC ( blue ) ductal clusters. The clusters in each group are combined. ns, p > 0.05; ∗ p < 0.05; student’s t test ( two-tailed ). MGAT3, beta-1,4-mannosyl-glycoprotein 4-beta-N-acetylglucosaminyltransferase; ns, not statistical; PDAC, pancreatic ductal adenocarcinoma; ST3GAL3, ST3 beta-galactoside alpha-2,3-sialyltransferase 3; ST6GAL1, ST6 beta-galactoside alpha-2,6-sialyltransferase 1; uMAP, Uniform Manifold Approximation and Projection.

Article Snippet: The slide was then incubated with anti-human ST6GAL1 (Proteintech, cat # 14355-1-AP) followed by Opal Polymer horseradish peroxidase Ms + Rb (Akoya Biosciences, cat # ARH1001EA) and tyramide-linked 650 Opal fluorophore (Akoya Biosciences, cat # FP1496001KT) to amplify signals.

Techniques: Transferring, Isolation, Sequencing, Comparison, Two Tailed Test

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Integrated Systems Analysis of the Murine and Human Pancreatic Cancer Glycomes Reveals a Tumor-Promoting Role for ST6GAL1

doi: 10.1016/j.mcpro.2021.100160

Figure Lengend Snippet: Profiling of ST6GAL1 and SNA in human pancreatic cancer shows association with the stage and survival. A , H&E of the normal pancreas ( left ), stage I pancreatic adenocarcinoma ( center ), and stage IV PDAC ( right ) stained from a BioMax human tissue microarray. B , multiplex OPAL IF staining of SNA ( yellow ), ST6GAL1 ( red ), and DAPI ( blue ) on the corresponding normal pancreas and stage I and stage IV pancreatic adenocarcinoma purchased from BioMax human tissue microarray. The scale bars represent 25 μm. C , quantification of SNA-positive cells per high-powered field based on multiplex IF in normal versus all cancerous cases in human tissue microarray. Owing to the high number of positive cells, each HPF was given a score (1–3) based on the number of SNA-positive cells per field. D , quantification of ST6GAL1-positive cells per high-powered field in normal versus all cancerous cases in human tissue microarray. E , distribution of the total ST6GAL1-positive cells per high-powered field across normal and each stage of pancreatic cancer based on multiplex IF imaging of human pancreatic cancer tissue microarray. Statistical significance was evaluated using the Student’s t test ( two-tailed ): ns, p > 0.05; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001. HFP, high-powered field; ns, not statistical; PDAC, pancreatic ductal adenocarcinoma; SNA, Sambucus nigra agglutinin; ST6GAL1, ST6 beta-galactoside alpha-2,6-sialyltransferase 1.

Article Snippet: The slide was then incubated with anti-human ST6GAL1 (Proteintech, cat # 14355-1-AP) followed by Opal Polymer horseradish peroxidase Ms + Rb (Akoya Biosciences, cat # ARH1001EA) and tyramide-linked 650 Opal fluorophore (Akoya Biosciences, cat # FP1496001KT) to amplify signals.

Techniques: Staining, Microarray, Multiplex Assay, Imaging, Two Tailed Test

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Integrated Systems Analysis of the Murine and Human Pancreatic Cancer Glycomes Reveals a Tumor-Promoting Role for ST6GAL1

doi: 10.1016/j.mcpro.2021.100160

Figure Lengend Snippet: Pancreas-specific deletion of ST6GAL1 reduces disease burden in murine PDAC. A , breeding schematic illustrating the generation of novel ST6KC mice. The offspring of parental strains crossed into p48-Cre mice drive the induction of mutant KRAS G12D and deletion of ST6GAL1 under the same promoter. B , IHC staining for ST6GAL1 ( brown ) in KC and ST6KC mice (n = 3 per group). The number of ST6GAL1+ cells per high-powered field is quantified. The scale bar represents 50 μm. C , IF staining for SNA ( yellow ) and DAPI ( blue ) in KC and ST6KC mice (n = 3 per group). The number of SNA+ cells per high-powered field is quantified. The scale bar represents 100 μm. D , H&E of 14-week-old FFPE pancreata from KC and ST6KC mice (n = 5 per group). The percent of the preserved normal pancreas area is quantified per high-powered field. The scale bar represents 200 μm. E , trichrome and gomori ( blue ) stain of FFPE pancreata from 14-week-old KC and ST6KC mice (n = 5 per group). The percent of collagen deposition fibrosis is quantified per high-powered field on the right. The scale bar represents 200 μm. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; Student’s t test ( two-tailed ). FFPE, formalin-fixed paraffin-embedded; IF, immunofluorescence; IHC, immunohistochemical; PDAC, pancreatic ductal adenocarcinoma; SNA, Sambucus nigra agglutinin; ST6GAL1, ST6 beta-galactoside alpha-2,6-sialyltransferase 1; ST6KC, ST6GAL1 flx/flx ;p48 Cre ; LSL KRASG12D .

Article Snippet: The slide was then incubated with anti-human ST6GAL1 (Proteintech, cat # 14355-1-AP) followed by Opal Polymer horseradish peroxidase Ms + Rb (Akoya Biosciences, cat # ARH1001EA) and tyramide-linked 650 Opal fluorophore (Akoya Biosciences, cat # FP1496001KT) to amplify signals.

Techniques: Mutagenesis, Immunohistochemistry, Staining, Two Tailed Test, Formalin-fixed Paraffin-Embedded, Immunofluorescence, Immunohistochemical staining

Journal: Cell reports

Article Title: Distinct RORγt-dependent Th17 immune responses are required for autoimmune pathogenesis and protection against bacterial infection

doi: 10.1016/j.celrep.2024.114951

Figure Lengend Snippet: (A–E) RNA-seq analysis of WT and RORγt K256R/K256R CD4 + T cells polarized under Th17 conditions. (A) The number of differentially expressed genes (DEGs, black) including upregulated (red) and downregulated (blue) genes with a cutoff at p < 0.05 and fold change (FC) >1.9. (B) Volcano plot shows DEGs between indicated Th17 cells (log 10 p on y axis and log 2 FC on x axis). Top downregulated (left) and upregulated (right) candidates in RORγt K256R/K256R cells are indicated. Red font: the top three downregulated candidates. (C) A Venn diagram illustrates the overlapping 32 genes between 223 pathogenic Th17-specific genes and 746 downregulated genes in RORγt K256R/K256R Th17 cells (GEO: GSE39820). , , , (D) Heatmap of the 32 overlapping genes described in (C). (E) qPCR analysis of relative mRNA levels of Lgals3 in Th0 and Th17 cells ( n = 4). (F and G) qPCR analysis of Lgals3 mRNA levels (F) and flow cytometric analysis of Lgals3 protein (G, left panels) and percentage (G, right panel) of Lgals3 + cells among indicated CD4 + T cells polarized under Th17 conditions ( n = 6). (H and I) Representative flow cytometric analysis (H) and percentage (I) of Lgals3 + cells among CD4 + T cells from the spleens of indicated untreated mice (left two panels) or from the CNS of indicated EAE-induced mice (right two panels) as described in ( n = 7). (J) Representative flow cytometric analysis (left three panels) and percentage of Lgals3 + cells (right panel) among CD4 + T cells in the colons of C. rodentium -infected indicated mice described in . Lgals3 −/− group is a negative staining control. (K) Mean EAE clinical scores at different days of Rag1 −/− mice adoptively transferred with sorted 1 × 10 5 indicated CD4 + GFP + T cells retrovirally expressing GFP alone (EV) or with Ccr6 and polarized under Th17 conditions ( n = 8, two independent experiments). (L) Number of CD4 + T cells recovered from the CNS of EAE-induced mice described in (K). (M) Mean EAE clinical scores of Rag1 −/− -recipient mice adoptively transferred with sorted 1 × 10 5 Tg TCR2D GFP + CD4 + T cells retrovirally expressing GFP together with scrambled short hairpin RNA (shRNA) or shRNA targeting Lgals3 (shLgals3) and polarized under Th17 conditions ( n = 6, two independent experiments). (N) Number of CD4 + T cells recovered from the CNS of EAE-induced mice described in (M). Data are presented as mean ± SEM. Statistical significance is indicated as **p < 0.05; ***p < 0.001; ns, not significant (two-tailed unpaired Student’s t test). Also see .

Article Snippet: Lgals3 antibodies (M3/38) were obtained from Miltenyi Biotec for flow cytometric analysis or Santa Cruz Biotechnology for the Western blotting.

Techniques: RNA Sequencing, Infection, Negative Staining, Control, Expressing, shRNA, Two Tailed Test

Journal: Cell reports

Article Title: Distinct RORγt-dependent Th17 immune responses are required for autoimmune pathogenesis and protection against bacterial infection

doi: 10.1016/j.celrep.2024.114951

Figure Lengend Snippet: (A) Representative flow cytometric analysis of IL-17A expression in WT and Lgals3 −/− CD4 + T cells polarized under Th17 conditions in vitro for 3 days ( n = 4). (B) Mean clinical score of indicated mice on different days after EAE induction ( n = 6, two independent experiments). (C) The number of CD4 + T cells infiltrated into the CNS of mice described in (B). (D) Mean clinical score of Rag1 −/− recipients adoptively transferred with 3 × 10 6 naive CD4 + T cells from indicated mice, followed by induction of EAE with MOG 35–55 ( n = 7–8, two independent experiments). (E) The number of CD4 + T cells infiltrated into the CNS of Rag1 −/− mice described in (D). (F) Body weight of indicated mice on different days after oral infection with 2 × 10 9 C. rodentium ( n = 8–9, two independent experiments). (G–I) Bacterial load (G), colon length (H), and the number of CD4 + T cells in the colons (I) of mice described in (F) at day 21 post infection. (J) Representative flow cytometric analysis (left panels) and percentage (right panel) of IL-17A + cells among CD4 + T cells recovered from the colons of indicated mice shown in (F). Data are presented as mean ± SEM. Statistical significance is indicated as *p < 0.01; **p < 0.05; ***p < 0.001; ns, not significant (two-tailed unpaired Student’s t test). Also see .

Article Snippet: Lgals3 antibodies (M3/38) were obtained from Miltenyi Biotec for flow cytometric analysis or Santa Cruz Biotechnology for the Western blotting.

Techniques: Expressing, In Vitro, Infection, Two Tailed Test

Journal: Cell reports

Article Title: Distinct RORγt-dependent Th17 immune responses are required for autoimmune pathogenesis and protection against bacterial infection

doi: 10.1016/j.celrep.2024.114951

Figure Lengend Snippet: (A) Scheme of potential Runx1 DNA-binding sites. Region 1 (Rgn1) and Rgn2 are two conserved Runx1-binding sites identified from ChIP-seq for Runx1 (GEO: GSE158093). E, exon. (B) ChIP signals with anti-FLAG antibody in in vitro differentiated WT Th17 cells retrovirally transduced with GFP alone (EV) or with FLAG-tagged Runx1 ( n = 4). (C) Schematic representation of indicated luciferase reporter constructs. P, Lgals3 promoter; I, Lgals3 intron; TK, minimal thymidine kinase gene promoter; Δ, deletion; Luc, luciferase. (D and E) Relative luciferase activity from indicated reporter shown in (C) transfected into 293T cells together with expression plasmid for Runx1 or control empty plasmid (Ctrl). Basic is a promoterless reporter ( n = 4). (F and G) Flow cytometric analysis of Lgal3 expression (left panels) and percentage of Lgals3 + cells among Cas9-expressing CD4 + T cells retrovirally transduced with nontargeting (NonT) sgRNA controls or sgRNA targeting to delete Rgn1 (F) or Rgn2 (G) and polarized under Th17 conditions ( n = 4). Gray: controls unstained (Unst) with the Lgals3 antibody. (H) Number of CD4 + T cells infiltrating the CNS of Rag1 −/− adoptively transferred with Tg Tcr2D Th17 cells retrovirally transduced with NonT, sgRgn1, or sgRgn2 followed by induction of EAE with MOG 35–55 ( n = 5). (I) The endpoint clinical score of mice as described in (H). (J) Flow cytometric analysis of Lgals3 in indicated CD4 + T cells expressing GFP alone or together with Runx1. Data are presented as mean ± SEM. Statistical significance is indicated as *p < 0.01; **p < 0.05; ***p < 0.001; ns, not significant (two-tailed unpaired Student’s t test). Also see .

Article Snippet: Lgals3 antibodies (M3/38) were obtained from Miltenyi Biotec for flow cytometric analysis or Santa Cruz Biotechnology for the Western blotting.

Techniques: Binding Assay, ChIP-sequencing, In Vitro, Transduction, Luciferase, Construct, Activity Assay, Transfection, Expressing, Plasmid Preparation, Control, Two Tailed Test

Journal: Cell reports

Article Title: Distinct RORγt-dependent Th17 immune responses are required for autoimmune pathogenesis and protection against bacterial infection

doi: 10.1016/j.celrep.2024.114951

Figure Lengend Snippet: (A) Representative flow cytometric analysis (left panels) and percentage (right panel) of Ccr6 + cells among indicated CD4 + T cells polarized under Th17 conditions for 3 days ( n = 6). (B) The number (top panels) and percentage (bottom panels) of indicated types of cells in the CNS of indicated mice immunized with MOG 35–55 . (C) Representative flow cytometric analysis (top panels), percentage (two bottom-left panels), and number (two bottom-right panels) of monocytes/macrophages (Mono/Mac) and neutrophils in the CNS of Rag1 −/− mice adoptively transferred with indicated CD4 + GFP + T cells retrovirally expressing GFP alone (EV) or with Lgals3 and polarized under Th17 conditions, followed by EAE induction as described in . (D) Representative flow cytometric analysis (left panels) and percentage (right panel) of BMDMs migrated to the bottom wells containing indicated CD4 + GFP + T cells retrovirally expressing GFP alone (EV) or with Lgals3 and differentiated under Th17 cells conditions in a Transwell migration assay ( n = 4 independent experiments). (E) Immunoblot (IB) analysis of FLAG-Lgals3 immunoprecipitated (IP) from the supernatants of CD4 + T cells transduced with empty retrovirus (EV) or virus expressing 3xFLAG-Lgals3 and polarized under Th17 condition for 3 days ( n = 4). Bottom two lanes are the immunoblot analysis of Lgals3 in the whole-cell lysates (WCLs) with anti-FLAG or anti-Lgals3 antibody. (F) Representative flow cytometric analysis (left panels) and percentage (right panel) of Ccr6 + cells among in vitro differentiated RORγt K256R/K256R Th17 cells co-cultured without (None) or with BMDMs for 18 h ( n = 4). (G) Representative flow cytometric analysis (left panels) and percentage (right panel) of Ccr6 + cells among CD4 + T cells recovered from the CNS of Rag1 −/− mice adoptively transferred with indicated CD4 + GFP + T cells retrovirally expressing GFP alone (EV) or with Lgals3 and polarized under Th17 conditions followed by EAE induction as described in ( n = 7–8). Analysis was conducted on day 10 post immunization. (H) Percentage of Ccr6 + cells among CD4 + T cells infiltrated into the CNS of WT or Lgals3 −/− mice immunized with MOG 35–55 as described in ( n = 6). (I) Percentage of Ccr6 + cells among in vitro differentiated Th17 cells co-cultured without (None) or with BMDMs for 18 h in the absence or presence of indicated neutralizing antibodies, analyzed by flow cytometry ( n = 4). (J) Percentage of Ccr6 + cells among in vitro differentiated RORγt K256R/K256R Th17 cells treated with vehicle or recombinant IL-1β ( n = 5). (K) Representative flow cytometric analysis (left panels) and percentage (right panel) of IL-1β + cells among Mono/Mac cells in the spleens (left two panels) or the CNS (middle two panels) of indicated mice immunized with MOG 35–55 (n = 8–9). (L) Number of IL-1β + cells among lymphocytes recovered from CNS of indicated EAE-induced mice. Data are presented as mean ± SEM. Statistical significance is indicated as *p < 0.01; **p < 0.05; ***p < 0.001; ns, not significant (two-tailed unpaired Student’s t test). Also see .

Article Snippet: Lgals3 antibodies (M3/38) were obtained from Miltenyi Biotec for flow cytometric analysis or Santa Cruz Biotechnology for the Western blotting.

Techniques: Expressing, Transwell Migration Assay, Western Blot, Immunoprecipitation, Transduction, Virus, In Vitro, Cell Culture, Flow Cytometry, Recombinant, Two Tailed Test

Journal: Cell reports

Article Title: Distinct RORγt-dependent Th17 immune responses are required for autoimmune pathogenesis and protection against bacterial infection

doi: 10.1016/j.celrep.2024.114951

Figure Lengend Snippet:

Article Snippet: Lgals3 antibodies (M3/38) were obtained from Miltenyi Biotec for flow cytometric analysis or Santa Cruz Biotechnology for the Western blotting.

Techniques: Virus, Recombinant, Modification, Protease Inhibitor, Lysis, Radio Immunoprecipitation, Isolation, cDNA Synthesis, SYBR Green Assay, Emulsion, Reporter Assay, Microarray, CRISPR, Luciferase, Control, Software

Journal: Journal of Inflammation Research

Article Title: Tumor-Infiltrating PD-L1+ Neutrophils Induced by GM-CSF Suppress T Cell Function in Laryngeal Squamous Cell Carcinoma and Predict Unfavorable Prognosis

doi: 10.2147/jir.s347777

Figure Lengend Snippet: Figure 1 CD66b+ neutrophils were predominantly located within the peritumoral stroma region in LSCC patients, which were associated with poorer prognosis. (A) Representative images were shown for CD45 and CD66b IHC staining of tumor tissues and normal tissues from LSCC patients. (B) Representative LSCC tumor tissues exhibited various density of CD66b+ neutrophils in peritumoral stroma and intratumoral regions. Bars = 50μm. (C) Infiltrating CD66b+ neutrophils in intratumoral and peritumoral region of tumor tissue, and epithelium and sub-epithelium region of normal tissues were analyzed. (D and E) LSCC patients with advanced T stage had more intratumoral and peritumoral infiltrated CD66b+ neutrophils. (F–I) Kaplan–Meier curves for overall survival and disease-free survival of 45 LSCC patients according to different density of CD66b+ neutrophils infiltrated in peritumoral stroma and intratumoral regions of tumor tissues. 50X, 200X, 400X: magnification of 50, 200, and 400 fold; *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Immunohistochemistry (IHC) Staining of Tissue Microarray Formalin-fixed and paraffin-embedded tumor tissues and matched normal mucosa tissues from the first LSCC patient cohort mentioned above were prepared for tissue microarray (TMA) construction as previously described.14 The TMA slides were incubated overnight at 4°C in wet box, with primary mouse anti-human CD45 antibody (60,287, Clone 4E9B2, Proteintech, Rosemont, USA), mouse anti-human CD66b antibody (392,902, Clone 6/40c, Biolegend, San Diego, USA) or rabbit polyclonal anti-human GM-CSF antibody (ab9741, Abcam, Cambridge, USA), followed with the incubation of biotin-conjugated secondary goat anti-mouse/rabbit IgG antibody at room temperature for one hour.

Techniques: Immunohistochemistry

Journal: Journal of Inflammation Research

Article Title: Tumor-Infiltrating PD-L1+ Neutrophils Induced by GM-CSF Suppress T Cell Function in Laryngeal Squamous Cell Carcinoma and Predict Unfavorable Prognosis

doi: 10.2147/jir.s347777

Figure Lengend Snippet: Figure 2 Immune cell infiltration profiles in LSCC. (A) Representative polychromatic dot plots displayed CD45+ leukocytes, CD3+ T cells, CD4+ T cells, CD8+ T cells γδT cells (γδTCR+) and neutrophils (CD66b+) in peripheral blood, tumor tissues and normal tissues from LSCC patients or healthy controls. (B) Calculative distribution of lymphocytes, neutrophils, NLR, CD3+T cells, CD4+T cells, CD8+T cells, γδT cells, and CD4+T cells /CD8 + T cells ratio in peripheral blood, tumor tissues and normal tissues. (C) Representative PD-1 expression on CD4+T/CD8+T/γδT cells and PDL-1 expression on CD66b+ neutrophils from the peripheral blood, tumor tissues and normal tissues. (D) Statistics analysis of PD-1+ CD4+T, PD-1+ CD8+T, PD-1+ γδT cells and PDL-1+ CD66b+ neutrophils. (E) Association analysis of TANs, NLR, CD3+ T cells or CD8+ T cells and tumor staging. (F) Linear correlation between TANs and CD3+ T cells, CD4+T cells, CD8+ T cells, or γδT cells. (G) Linear correlation between PD-L1+ TANs and PD-1+ CD4+ T cells, PD-1+ CD8+ T cells, or PD-1+ γδT cells. Each dot represents an independent data point as determined by flow cytometry. ns = p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Immunohistochemistry (IHC) Staining of Tissue Microarray Formalin-fixed and paraffin-embedded tumor tissues and matched normal mucosa tissues from the first LSCC patient cohort mentioned above were prepared for tissue microarray (TMA) construction as previously described.14 The TMA slides were incubated overnight at 4°C in wet box, with primary mouse anti-human CD45 antibody (60,287, Clone 4E9B2, Proteintech, Rosemont, USA), mouse anti-human CD66b antibody (392,902, Clone 6/40c, Biolegend, San Diego, USA) or rabbit polyclonal anti-human GM-CSF antibody (ab9741, Abcam, Cambridge, USA), followed with the incubation of biotin-conjugated secondary goat anti-mouse/rabbit IgG antibody at room temperature for one hour.

Techniques: Expressing, Flow Cytometry

Journal: Oncogene

Article Title: Transcriptome profiling of a TGF-beta-induced epithelial-to-mesenchymal transition reveals extracellular clusterin as a target for therapeutic antibodies.

doi: 10.1038/onc.2009.399

Figure Lengend Snippet: Figure 2 Validation of clusterin upregulation by semi-quantitative reverse transcription–PCR, western blot analysis and immunofluorescence microscopy. (a) Semi-quantitative reverse transcription–PCR analysis (bottom panels) of the clusterin transcript (CLU) in BRI-JM01 cells induced after 2, 4, 6 and 24 h of TGF-b1 (100 pM) exposure confirming the clusterin transcript modulation observed in the microarray experiments (The graph underneath represents normalized data obtained from the microarray experiments. Bars represent the t-test P-value of 4–6 experiments performed for each time point.) The Eef1a-1 transcript (top panel), which does not change upon TGF-b1 treatment, was used as loading control. (b) Western blot analysis of whole-cell lysates (WCL) from BRI-JM01 cells grown in the absence or presence of 100 pM TGF-b1 (24 h) confirming the upregulation of clusterin and showing the uncleaved precursor form (pCLU) of clusterin in the WCL, and the the mature processed form (sCLU) in both the WCL and the CM. Membranes containing the WCL samples were reprobed for b-actin to confirm equal loading. (c) Immunofluorescence microscopy (magnification 1000) of BRI-JM01 cells treated with 100 pM TGF-b1 (24 h) show clusterin to be close to the cell’s outer membrane (top panels) and (d) colocalized with the Golgi marker b-COP (bottom) (red, clusterin; green, b-COP; blue, diamidino phenylindole (DAPI)-stained nuclei; magnification 400). (e) Western blot of the CM confirming the increased levels of secreted clusterin (sCLU) in the medium of BRI-JM01 cells treated with 100 pM TGF-b1 (24 h). CM, conditioned medium; sCLU, secreted clusterin; TGF-b, transforming growth factor-b.

Article Snippet: Antibodies against the following proteins were purchased: clusterin (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), E-cadherin (Sigma, Minneapolis, MN, USA; R&D Systems, Oakville, Canada), ZO-1 (anti-ZO1; Chemicon, Billerica, MA, USA; Zymed, San Francisco, CA, USA), b-COP (anti-b-COP; Cedarlane, Burlington, Canada) smad2 and phospho-smad2 (both Cell Signaling Technology, Danvers, MA, USA) and b-actin (Sigma).

Techniques: Biomarker Discovery, Reverse Transcription, Western Blot, Microscopy, Microarray, Control, Membrane, Marker, Staining

Journal: Oncogene

Article Title: Transcriptome profiling of a TGF-beta-induced epithelial-to-mesenchymal transition reveals extracellular clusterin as a target for therapeutic antibodies.

doi: 10.1038/onc.2009.399

Figure Lengend Snippet: Figure 3 Antibodies against sCLU block the TGF-b1-induced loss of junctional ZO-1 and E-cadherin in BRI-JM01 cells. Immunofluorescence microscopy of ZO-1 in BRI-JM01 cells grown (a) in the absence or presence of 100 pM TGF-b1 with or without antibodies against TGF-b (anti-TGF-b, 10 nM) or clusterin (anti-clu, 8 mg/ml), or (b) in CM from untreated BRI-JM01 cells (CM-CTL) or from cells treated with 100 pM TGF-b1 for 24 h (CM-TGF-b1) both in the absence or presence of antibodies against (anti-TGF-b, 10 nM) or clusterin (anti-clu, 8 mg/ml). For all the panels: red, ZO-1; blue, diamidino phenylindole (DAPI)-stained nuclei (magnification: 400). (c) Flow cytometric evaluation of the cell-surface levels of E-cadherin expressed by BRI-JM01 cells exposed to TGF-b1 (100 pM) in the absence or presence of clusterin polyclonal IgG (anti-clu, 8 mg/ml). Cell populations expressing high ( þ ) and low () levels of cell-surface E-cadherin are indicated. CM, conditioned medium; sCLU, secreted clusterin; TGF-b, transforming growth factor-b; ZO, zona occludens.

Article Snippet: Antibodies against the following proteins were purchased: clusterin (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), E-cadherin (Sigma, Minneapolis, MN, USA; R&D Systems, Oakville, Canada), ZO-1 (anti-ZO1; Chemicon, Billerica, MA, USA; Zymed, San Francisco, CA, USA), b-COP (anti-b-COP; Cedarlane, Burlington, Canada) smad2 and phospho-smad2 (both Cell Signaling Technology, Danvers, MA, USA) and b-actin (Sigma).

Techniques: Blocking Assay, Microscopy, Staining, Expressing

Journal: Oncogene

Article Title: Transcriptome profiling of a TGF-beta-induced epithelial-to-mesenchymal transition reveals extracellular clusterin as a target for therapeutic antibodies.

doi: 10.1038/onc.2009.399

Figure Lengend Snippet: Figure 4 Purified clusterin promotes a spindle-shaped morphol- ogy and loss of junctional ZO-1 in BRI-JM01 cells. (a) Coomassie blue-stained gel under non-reducing (DTT (dithiothreitol)) and reducing ( þ DTT) conditions of purified recombinant human clusterin (b) expressed and secreted by HEK-293-E6 cells. (b) BRI- JM01 cells treated with 200 nM purified clusterin (24 h) show a spindle-shaped morphology (top; magnification 40) and loss of tight-junctional ZO-1 (red, bottom; magnification 400). HEK, human embryonic kidney; ZO, zona occludens.

Article Snippet: Antibodies against the following proteins were purchased: clusterin (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), E-cadherin (Sigma, Minneapolis, MN, USA; R&D Systems, Oakville, Canada), ZO-1 (anti-ZO1; Chemicon, Billerica, MA, USA; Zymed, San Francisco, CA, USA), b-COP (anti-b-COP; Cedarlane, Burlington, Canada) smad2 and phospho-smad2 (both Cell Signaling Technology, Danvers, MA, USA) and b-actin (Sigma).

Techniques: Staining, Recombinant

Journal: Oncogene

Article Title: Transcriptome profiling of a TGF-beta-induced epithelial-to-mesenchymal transition reveals extracellular clusterin as a target for therapeutic antibodies.

doi: 10.1038/onc.2009.399

Figure Lengend Snippet: Figure 6 Clusterin polyclonal IgG inhibits the invasive behavior of cell lines other than the BRI-JM01 cell line. Clusterin polyclonal IgG (anti-clu, 8 mg/ml) inhibits (a) the stellate morphology of 4T1 and PC3 tumor cells when cultured in Matrigel for 3 weeks (magnification 40). (b) Western blot analysis confirming the presence of sCLU in the CM of 4T1, NMuMG and PC3 cells. (c) Clusterin polyclonal IgG (anti-clu, 8 mg/ml) inhibits the Matrigel invasion of 4T1, NMuMG and PC3 cell lines ing a Transwell invasion assay. Results (expressed relative to non-treated cells) are shown as the average (±s.d.) of two independent experiments. (d) Clusterin polyclonal IgG (anti-clu, 8 mg/ml) does not significantly affect the growth-inhibitory response induced by TGF-b1 in 4T1, NMuMG and PC3 cells. Results are expressed relative to non-treated cells and are shown as the average (±s.d.) of two independent experiments performed in triplicate. (e) Western blot analysis showing the presence of sCLU in the CM of MDA-MB231LM2 cells (left panel). Clusterin polyclonal IgG (anti-clu, 8 mg/ml) inhibits the Matrigel invasion of these cells in a Transwell assay (right panel). Results (expressed relative to non-treated cells) are shown as the average (±s.d.) of two independent experiments. CM, conditioned medium; sCLU, secreted clusterin; TGF-b, transforming growth factor-b.

Article Snippet: Antibodies against the following proteins were purchased: clusterin (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), E-cadherin (Sigma, Minneapolis, MN, USA; R&D Systems, Oakville, Canada), ZO-1 (anti-ZO1; Chemicon, Billerica, MA, USA; Zymed, San Francisco, CA, USA), b-COP (anti-b-COP; Cedarlane, Burlington, Canada) smad2 and phospho-smad2 (both Cell Signaling Technology, Danvers, MA, USA) and b-actin (Sigma).

Techniques: Cell Culture, Western Blot, Transwell Invasion Assay, Transwell Assay

Journal: Oncogene

Article Title: Transcriptome profiling of a TGF-beta-induced epithelial-to-mesenchymal transition reveals extracellular clusterin as a target for therapeutic antibodies.

doi: 10.1038/onc.2009.399

Figure Lengend Snippet: Figure 7 Clusterin monoclonal antibodies inhibit the motility of 4T1 cells and significantly reduce 4T1 cell metastasis to lungs after orthotopic implantation. (a) Evaluation of the ability of in-house-generated clusterin monoclonal antibodies 11E2, 16B5, 16C11 and 20G3 at a concentration of 8 mg/ml (black bars), and a commercially available clusterin monoclonal antibody B5 and anti-peptide polyclonal IgG, C18 (white bars), to block 4T1 cell motility in a BCSM assay. The average motility (±s.d.) was determined by measuring ink clearance in 10 independent microscopic fields (per treatment) and is expressed as average ink clearance/cell/24 h relative to non-treated control cells (gray bar). The hatched line depicts the cut-off that we used to define an antibody as having neutralizing activity, which was based on the degree of inhibition caused by B5 antibody. (b) Experimental design. 4T1 cells (4 104) were injected in the left # 4 inguinal mammary gland. Animals were treated (5 mg/kg) with neutralizing (16B5, 16C11 and 11E2) clusterin antibody, non-neutralizing (20G3) clusterin antibody or saline (control) thrice a week (intraperitoneally), starting the day of cell implantation. Mice were killed on day 28. (c) The number of macroscopically grossly visible lung metastasis in the mice were quantified 28 days post tumor cell implantation and statistically analysed using the non-parametric Mann–Whitney U-test (n ¼ 10 for the saline, 20G3 and 11E2 groups; n ¼ 9 for the 16B5 and 16C11 groups). BCSM, black cellular spreading and motility.

Article Snippet: Antibodies against the following proteins were purchased: clusterin (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), E-cadherin (Sigma, Minneapolis, MN, USA; R&D Systems, Oakville, Canada), ZO-1 (anti-ZO1; Chemicon, Billerica, MA, USA; Zymed, San Francisco, CA, USA), b-COP (anti-b-COP; Cedarlane, Burlington, Canada) smad2 and phospho-smad2 (both Cell Signaling Technology, Danvers, MA, USA) and b-actin (Sigma).

Techniques: Bioprocessing, Generated, Concentration Assay, Blocking Assay, Control, Activity Assay, Inhibition, Injection, Saline, MANN-WHITNEY

Journal: PLoS ONE

Article Title: Mesenchymal stromal cell-derived extracellular vesicles reduce lung inflammation and damage in nonclinical acute lung injury: Implications for COVID-19

doi: 10.1371/journal.pone.0259732

Figure Lengend Snippet: (A) Antibody microarray particle analysis of tetraspanin proteins. (B) Electrochemiluminescent multiplexed immunoassay detection of tetraspanin proteins. (C) Western blot analysis of MSC-EV proteins flotillin-1, Annexin-2, Syntenin-1, MHC-I and contaminating proteins MHC-II and calreticulin. (D) Immunogold cryo-transmission electron microscopy imaging for CD63 and phosphatidylserine. Atomic force microscopy for MSC-EV morphology and size. (E) Nanoparticle tracking for MSC-EV size and concentration. (F) MSC-EV particle density correlated to phospholipid content. Error bars are SEM of the mean. *p ≤0.05 vs CD63. Scale bar denotes 100 nm.

Article Snippet: Lysates were volume loaded and separated on a 12% Bolt Bis-Tris Gel and probed using Abcam antibodies against flotillin-1 (ab133497 at 1:10,000 dilution), Annexin-2 (ab41803 at 1:1000 dilution), Syntenin-1 (ab19903 at 1:1000 dilution), MHC-I (ab110645 at 1:1000 dilution), MHC-II (157210 at 1:10000 dilution), and Calreticulin (ab92516 at 1:1000 dilution).

Techniques: Microarray, Western Blot, Transmission Assay, Electron Microscopy, Imaging, Microscopy, Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: Capability of Human Dendritic Cells Pulsed with Autologous Induced Pluripotent Stem Cell Lysate to Induce Cytotoxic T Lymphocytes against HLA-A33-Matched Cancer Cells

doi: 10.3390/ijms232112992

Figure Lengend Snippet: Heatmap analysis of gene expression of iPSCs, SW48 and T47D cells, and fibroblasts. The iPSC data were obtained from microarray analysis. The SW48 and T47D cell and fibroblast data were obtained from the NCBI GEO database. The listed genes are highly expressed in iPSCs and are very lowly or not expressed in fibroblasts (gene expression level < 5). The change from red to light purple indicates the gene expression level changing from 15 to 0. The 99 genes in the list were selected in order of highest gene expression in the iPSCs.

Article Snippet: The SW48 (colorectal cancer: HLA-A33), T47D (breast cancer: HLA-A33), and T98G (glioblastoma: HLA-A02) cell lines were obtained from American Type Culture Collection (Manassas, VA, USA).

Techniques: Gene Expression, Microarray

Journal: International Journal of Molecular Sciences

Article Title: Capability of Human Dendritic Cells Pulsed with Autologous Induced Pluripotent Stem Cell Lysate to Induce Cytotoxic T Lymphocytes against HLA-A33-Matched Cancer Cells

doi: 10.3390/ijms232112992

Figure Lengend Snippet: TAAs selected by the NCI Pilot Project. The list shows the ranking of TAAs selected as described previously . The iPSC data were obtained using microarray analysis. The iPSC, SW48 and T47D cell, and fibroblast data were obtained from the NCBI GEO database. The change from red to light purple indicates the gene expression level changing from 15 to 0.

Article Snippet: The SW48 (colorectal cancer: HLA-A33), T47D (breast cancer: HLA-A33), and T98G (glioblastoma: HLA-A02) cell lines were obtained from American Type Culture Collection (Manassas, VA, USA).

Techniques: Microarray, Gene Expression