inflamm cell signal  (Cell Signaling Technology Inc)

Journal: PLOS One

Article Title: Consequences of platelet-educated cancer cells on the expression of inflammatory and metastatic glycoproteins

doi: 10.1371/journal.pone.0317096

Figure Lengend Snippet: (A) Graph shows the mean fold change (percentage) of mRNA expression of 43 GT families in pancreatic cancer cells (PANC-1, MIA PaCa-2, Capan-2) after 3 hours of interaction of cancer cells with platelets compared to before their interaction (control). Colors represent an overexpression of at least 150% (red), 100% (pink), 50% (purple) or less than 50% (blue) of the mRNA of GT families in cancer cells after platelet interaction or, conversely, an underexpression of at least 25% (red), 15% (pink), 10% (purple) or less than 10% (blue). Data were normalized to the housekeeping gene HPRT1 and histogram represents the mean of three independent experiments (ANOVA test, ****P < 0.0001, * P = 0.0401). (B) Representative images and corresponding quantification of glycan motif detection using 4 lectins: VVL/VVA, (left upper panel) AAL (right upper panel), ConA (left lower panel), PHA-E (right lower panel) by Western Blot of pancreatic cancer cell lysates (PANC-1, MIA PaCa-2 or Capan-2, 40 µg) interacting (+) or not (−) with platelets for 3 hours. GAPDH expression was detected as a loading control for the experiment. The graphs show the mean of integrated density (+/− SEM) of glycan motif expression normalized to cancer cells alone (t-test, **P = 0.0086, * P = 0.0240, ns = not significant). (C) The graph represents the mean fold change (percentage) of mRNA expression of 124 genes encoding proteins involved in inflammation and metastasis in a human colorectal cancer cell line (HT-29) after 3 hours of interaction with platelets compared to before their interaction. Colors represent an overexpression of at least 300% (red), 200% (pink), 100% (purple) or less than 100% (blue) of the mRNA in platelet-educated cancer cells. Data were normalized to the housekeeping gene HPRT1. (D) Protein-protein interaction network using STRING database 12.0. Only the 38 genes with at least an 100% overexpression were considered to perform this analysis. Nodes represent proteins and each node color (red, yellow, dark blue, light blue or green) indicates a gene cluster. Edges represent known interactions from the curated database (light blue) or experimentally determined (pink); scientific literature (yellow/green); co-expression (black) or protein homology (dark blue). (E) Representative image of fibronectin (FN1, 273 kDa, left panel) detection by Western Blot in 15 µg of PANCO2 cell lysates 6, 24 or 48 hours after their interaction with platelets (+) or not (−). GAPDH expression was detected as a loading control. The histogram (down panel) presents the relative quantification of the FN1/GAPDH ratio in PANCO2 cells + platelets, normalized to PANCO2 cells alone, over time up to 48 hours.

Article Snippet: FN1 , Fibronectin 1 , 166 , Cell adhesion, Wound healing, Tissue development, Cell migration, Angiogenesis, Inflammation, Cell signaling (proliferation, survival, gene expression), Stem cell differentiation, Support of tissue and organs, Cancer metastasis , Breast, lung, colorectal, ovarian, prostate, melanoma, pancreatic, gastric and liver.

Techniques: Expressing, Control, Over Expression, Glycoproteomics, Western Blot, Quantitative Proteomics

Journal: PLOS One

Article Title: Consequences of platelet-educated cancer cells on the expression of inflammatory and metastatic glycoproteins

doi: 10.1371/journal.pone.0317096

Figure Lengend Snippet: Summary of genes over-expressed by at least 100% in platelet-educated cancer cells compared to cancer cells alone.

Article Snippet: FN1 , Fibronectin 1 , 166 , Cell adhesion, Wound healing, Tissue development, Cell migration, Angiogenesis, Inflammation, Cell signaling (proliferation, survival, gene expression), Stem cell differentiation, Support of tissue and organs, Cancer metastasis , Breast, lung, colorectal, ovarian, prostate, melanoma, pancreatic, gastric and liver.

Techniques: Over Expression, Protease Inhibitor, Activity Assay, Binding Assay, Migration, Gene Expression, Cell Differentiation, Membrane, Infection, Transduction, Activation Assay, Transplantation Assay, Coagulation

Journal: PLOS One

Article Title: Consequences of platelet-educated cancer cells on the expression of inflammatory and metastatic glycoproteins

doi: 10.1371/journal.pone.0317096

Figure Lengend Snippet: (A) Using miRNA transfection, we generated a murine pancreatic cancer line that underexpress fibronectin, called PANCO2 low FN1 cells. (B) Representative graphs and corresponding quantification of GFP detection (upper panels) by flow cytometry in PANCO2 mock (brown) vs. PANCO2 low FN1 (blue) cells. The histogram represents the mean value of four independent experiments (t-test, ****P < 0.0001). Representative graphs and respective quantification of FN1 detection (lower panels) with unconjugated anti-mouse FN1 antibody (2 µg/mL) and AF647-conjugated anti-rabbit IgG secondary antibody (2 µg/mL) by flow cytometry in PANCO2 mock (brown) vs. PANCO2 low FN1 (blue) cells compared to isotype control (2 µg/mL) detected with AF47-conjugated anti-rabbit IgG secondary antibody (brown curves). Histogram shows mean fluorescence intensity (+/− SEM) from three independent experiments (t-test, **P = 0.0025). (C) Representative images of three independent experiments of PANCO2 mock or PANCO2 low FN1 cells over time up to 24 hours (holotomographic microscopy bars, 10 µm). (D) Curves (left panel) were generated with the “Cell Death Assay” module showing the percentage of cell death in PANCO2 mock (solid line) or PANCO2 low FN1 (dashed line) cells over 24 hours. The histogram (right panel) shows the mean area under the preceding curve (+/- SEM) of three independent experiments (t-test, ns = not significant). (E) Representative images (left panel) of nine independent experiments of wound healing assay experiments evaluating PANCO2 mock or PANCO2 low FN1 cell migration over 24 hours (10X objective, bars 125 µm). Graph (right panel) shows the mean percentage of area recovery (+/− SEM), from these nine independent experiments, corresponding to the migration of PANCO2 mock or PANCO2 low FN1 cells (5 images per experiment and per well, t-test, ****P < 0,0001). (F) Representative images (left panel) of five independent migration assays to detect PANCO2 mock or PANCO2 low FN1 migrated cells, with Hoechst 33342 after 16 hours. The negative control was performed with the same cells but without FCS in the lower chamber (20X objective, bars 125 µm). Graph (right panel) represents the mean (+/− SEM) of the number of migrated PANCO2 mock or PANCO2 low FN1 cells, from five independent experiments, after 16 hours of incubation (10 images per experiment and per well, t-test, * P = 0.03, n = 5).

Article Snippet: FN1 , Fibronectin 1 , 166 , Cell adhesion, Wound healing, Tissue development, Cell migration, Angiogenesis, Inflammation, Cell signaling (proliferation, survival, gene expression), Stem cell differentiation, Support of tissue and organs, Cancer metastasis , Breast, lung, colorectal, ovarian, prostate, melanoma, pancreatic, gastric and liver.

Techniques: Transfection, Generated, Flow Cytometry, Control, Fluorescence, Microscopy, Wound Healing Assay, Migration, Negative Control, Incubation

Journal: PLOS One

Article Title: Consequences of platelet-educated cancer cells on the expression of inflammatory and metastatic glycoproteins

doi: 10.1371/journal.pone.0317096

Figure Lengend Snippet: (A) Schematic diagram of the experimental procedure: platelets were allowed to interact with the cancer cells (cancer cell:platelet ratio = 1:50) for 3 hours. The interactions were stopped by extensively washing the cancer cells with PBS-/-. the cancer cells were recorded to detect any changes in cancer cell behavior for another 3, 21 or 45 hours. (B) Representative images of three independent experiments of PANCO2 mock or PANCO2 low FN1 cells, in the presence (+) or in absence (−) of platelets over time up to 24 hours (holotomographic microscopy bars, 10 µm). (C) The curves, generated using the Cell Death Assay module, represent the percentage of cell death in PANCO2 mock (solid line) or PANCO2 + platelets (solid line with circle) cells (upper panel) , and in PANCO2 low FN1 (dashed line) or PANCO2 low FN1 + platelets (solid line with triangle) cells (middle panel) over a 24-hour period. The histogram in the bottom panel shows the mean area under the preceding curves (+/− SEM) from three independent experiments, with or without platelets (+/−) (ANOVA, ns = not significant). (D) Representative images (left panel) from nine independent wound healing assay experiments evaluating the migration of PANCO2 mock or PANCO2 low FN1 cells in the presence (+) or absence (−) of platelets over 24 hours (10X objective, scale bars = 250 µm). The graph (right panel) shows the mean percentage of area recovery (+/− SEM) from these nine independent experiments, reflecting the migration of PANCO2 mock or PANCO2 low FN1 cells, with (+) or without (−) platelets (5 images per experiment and per well, ANOVA test, * P < 0.0188). (E) Representative image (left panel) of FN1 detection by Western Blot in 15 µg of PANCO2 low FN1 lysates 6, 24 or 48 hours after their interaction with platelets (+) or not (−). Detection of GAPDH was used as a loading control for the experiment. Graph (right panel) shows the relative quantification of the FN1/GAPDH ratio in PANCO2 low FN1 cells normalized to PANCO2 low FN1 cells alone over time up to 48 hours. (F) Evaluation of cytokine expression in PANCO2 mock (light blue) or PANCO2 low FN1 (brown) cells after 6 (left panel) or 24 hours (right panel) interaction with platelets, normalized to PANCO2 or PANCO2 low FN1 cells alone, respectively, over a period of up to 24 hours using a multiplex ELISA workflow. Graphs represent the mean (+/− SEM) of relative fluorescence unit per cell normalized to cancer cells alone from three independent experiments.

Article Snippet: FN1 , Fibronectin 1 , 166 , Cell adhesion, Wound healing, Tissue development, Cell migration, Angiogenesis, Inflammation, Cell signaling (proliferation, survival, gene expression), Stem cell differentiation, Support of tissue and organs, Cancer metastasis , Breast, lung, colorectal, ovarian, prostate, melanoma, pancreatic, gastric and liver.

Techniques: Microscopy, Generated, Wound Healing Assay, Migration, Western Blot, Control, Quantitative Proteomics, Expressing, Multiplex Assay, Enzyme-linked Immunosorbent Assay, Fluorescence