Journal: EMBO Molecular Medicine

Article Title: ENOblock synergizes with colistin to treat Acinetobacter baumannii infections

doi: 10.1038/s44321-025-00331-2

Figure Lengend Snippet: ( A ) Schematic of the bacterial adhesion/invasion assay. ( B ) Analysis of Ab ATCC 17978 and Ab CR17 strains adhesion into HeLa and macrophage cells with (1xMIC) and without ENOblock treatment. The data are presented as means of three biological replicates ± SEM, For HeLa cells, * P = 0.023: Ab ATCC 17978 vs Ab ATCC 17978 + ENOblock and * P = 0.048: Ab CR17 vs Ab CR17 + ENOblock (two-tailed Student’s t test). For macrophage cells, * P = 0.018: Ab ATCC 17978 vs Ab ATCC 17978 + ENOblock and * P = 0.035: Ab CR17 vs Ab CR17 + ENOblock (two-tailed Student’s t test). ( C ) Analysis of Ab ATCC 17978 and Ab CR17 strains invasion into HeLa and macrophage cells with (1×MIC) and without ENOblock treatment. The data are presented as means of three biological replicates ± SEM, For HeLa cells, * P = 0.001: Ab ATCC 17978 vs Ab ATCC 17978 + ENOblock and * P = 0.011: Ab CR17 vs Ab CR17 + ENOblock (two-tailed Student’s t test). For macrophage cells, * P = 0.001: Ab ATCC 17978 vs Ab ATCC 17978 + ENOblock and * P = 0.002: Ab CR17 vs Ab CR17 + ENOblock (two-tailed Student’s t test). ( D ) Immunostaining of fibronectin of HeLa cells (magenta) and Ab ATCC 17978 and Ab CR17 strains (green) pretreated with ENOblock (0× and 1×MIC), after bacterial adherence for 2 h, was performed by specific primary antibodies against both strains and their respective secondary antibodies. Blue staining shows the location of HeLa cell nuclei. A representative image out of three biological replicates is shown. .

Article Snippet: Rabbit anti-human fibronectin , Merck , Cat# F3648.

Techniques: Invasion Assay, Two Tailed Test, Immunostaining, Staining

Journal: EMBO Molecular Medicine

Article Title: ENOblock synergizes with colistin to treat Acinetobacter baumannii infections

doi: 10.1038/s44321-025-00331-2

Figure Lengend Snippet: ( A – C ) Consensus-spectrum (CIS) of enolase, plasminogen and ENOblock, enolase, fibronectin and ENOblock, and enolase, fibrinogen and ENOblock. ( D – F ) Structural models of protein-protein complex generated by Alphafold3 analysis by docking of ENOblock (ball and stick), enolase (pelorous ribbons) and plasminogen, fibronectin or fibrinogen (tangerine ribbons). The intermolecular interactions from the obtained complex were according to ISM method–detected interaction domains (yellow ribbons). ( G – I ) Inhibition of Ab adherence to immobilized plasminogen, fibronectin and fibrinogen by free enolase. Ab ATCC 17978 strain was incubated in plasminogen, fibronectin and fibrinogen-coated wells for 3 h at room temperature, containing increasing concentrations of free enolase (0, 10, 50 and 100 mg/L). The data are presented as means of three biological replicates ± SEM. For plasminogen, * P = 0.023: Ab ATCC 17978 vs Ab ATCC 17978 + enolase (10 mg/L), * P = 0.002: Ab ATCC 17978 vs Ab ATCC 17978 + enolase (50 mg/L) and * P = 0.001: Ab ATCC 17978 vs Ab ATCC 17978 + enolase (100 mg/L) (two-tailed Student’s t test). For fibronectin, * P = 0.006: Ab ATCC 17978 vs Ab ATCC 17978 + enolase (10 mg/L), * P = 0.025: Ab ATCC 17978 vs Ab ATCC 17978 + enolase (50 mg/L) and * P = 0.005: Ab ATCC 17978 vs Ab ATCC 17978 + enolase (100 mg/L) (two-tailed Student’s t test). For fibrinogen, * P = 0.039: Ab ATCC 17978 vs Ab ATCC 17978 + enolase (10 mg/L), * P = 0.017: Ab ATCC 17978 vs Ab ATCC 17978 + enolase (50 mg/L) and * P = 0.002: Ab ATCC 17978 vs Ab ATCC 17978 + enolase (100 mg/L) (two-tailed Student’s t test). ( J – L ) Inhibition of Ab adherence to immobilized plasminogen, fibronectin and fibrinogen by ENOblock. Ab ATCC 17978 strain was incubated in plasminogen, fibronectin and fibrinogen-coated wells for 2 h at room temperature, containing increasing concentrations of ENOblock (0.5× and 1×MIC). The data are presented as means of three biological replicates ± SEM, * P < 0.05: treatment vs no treatment; two-tailed Student’s t test. For plasminogen, * P = 0.039: Ab ATCC 17978 vs Ab ATCC 17978 + ENOblock (0.5×MIC) and * P = 0.008: Ab ATCC 17978 vs Ab ATCC 17978 + ENOblock (1xMIC) (two-tailed Student’s t test). For fibronectin, * P = 0.026: Ab ATCC 17978 vs Ab ATCC 17978 + ENOblock (0.5×MIC) and * P = 0.042: Ab ATCC 17978 vs Ab ATCC 17978 + ENOblock (1×MIC) (two-tailed Student’s t test). For fibrinogen, * P 0.022 = Ab ATCC 17978 vs Ab ATCC 17978 + ENOblock (0.5×MIC) and * P = 0.045: Ab ATCC 17978 vs Ab ATCC 17978 + ENOblock (1xMIC) (two-tailed Student’s t test). Adherent bacteria to plasminogen, fibronectin and fibrinogen-coated wells were quantified by serial dilutions as described in materials and methods. Results were expressed as the percentage of total untreated Ab adhered to immobilized plasminogen, fibronectin and fibrinogen. .

Article Snippet: Rabbit anti-human fibronectin , Merck , Cat# F3648.

Techniques: Generated, Inhibition, Incubation, Two Tailed Test, Bacteria

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: Primary antibodies used were directed against a human polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3, AF7174, Bio-techne, 0.5 μg/mL), a human fibronectin (FN1, 26836, Cell Signaling Technology, 1/1000) and a mouse FN1 (Ab199056, Abcam, 1/1000).

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: Primary antibodies used were directed against a human polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3, AF7174, Bio-techne, 0.5 μg/mL), a human fibronectin (FN1, 26836, Cell Signaling Technology, 1/1000) and a mouse FN1 (Ab199056, Abcam, 1/1000).

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: Primary antibodies used were directed against a human polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3, AF7174, Bio-techne, 0.5 μg/mL), a human fibronectin (FN1, 26836, Cell Signaling Technology, 1/1000) and a mouse FN1 (Ab199056, Abcam, 1/1000).

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

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Spatial transcriptomics reveals prognostically LYZ + fibroblasts and colocalization with FN1 + macrophages in diffuse large B-cell lymphoma

doi: 10.1007/s00262-025-03968-7

Figure Lengend Snippet: Prognostic value of LYZ and LILRB4 proteins in DLBCL receiving R-CHOP ( n = 37, 10X) and aNSCLC receiving ICIs ( n = 29, 2X and 10X). A Representative IHCs staining of LYZ in patient 1 (PFS = 1697 days) and patient 2 (PFS = 341 days), LILRB4 in patient 3 (PFS = 64 days) and patient 4 (PFS = 826 days). B Kaplan–Meier survival curves of PFS grouped by the LYZ and LILRB4 expression in DLBCL. C Representative mIF staining of LYZ, LILRB4, and pan Cytokeratin. D Kaplan–Meier survival curves of OS grouped by the LYZ and LILRB4 expression in aNSCLC. E Kaplan–Meier survival curves of PFS grouped by the COL3A1 expression and FN1 + macrophages in DLBCL. DLBCL: diffuse large B cell lymphoma; R-CHOP: rituximab, cyclophosphamide, doxorubicin: vincristine, and prednisone; aNSCLC: advanced non-small cell lung cancer; ICIs: immune checkpoint inhibitors; IHC: immunohistochemistry; PFS: progression-free survival; mIF: multiple immunofluorescence; and OS: overall survival

Article Snippet: Rabbit anti-human FN1 IgG antibody , #26836S , Cell Signaling Technology.

Techniques: Staining, Expressing, Immunohistochemistry, Immunofluorescence

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Spatial transcriptomics reveals prognostically LYZ + fibroblasts and colocalization with FN1 + macrophages in diffuse large B-cell lymphoma

doi: 10.1007/s00262-025-03968-7

Figure Lengend Snippet: Prognostic significance of autoantibodies against COL1A2, COL3A1, and FN1 in patients with DLBCL receiving R-CHOP ( n = 20 and 125) and NSCLC treated with ICIs ( n = 36). A Volcano plot, boxplot, and representative density plots of patients illustrating the distribution of COL1A2 autoantibodies in DLBCL. B Kaplan–Meier curve for PFS based on COL1A2 autoantibody levels in DLBCL. C Kaplan–Meier curves for OS and PFS in NSCLC patients receiving immunotherapy, stratified by COL1A2 and COL3A1 mRNA levels in GSE128989 and autoantibody presence, alongside representative density plots of COL1A2 and COL3A1 autoantibodies. D Comparison of FN1 autoantibody levels between healthy controls and DLBCL, with Kaplan–Meier analysis for PFS based on FN1 autoantibody levels. DLBCL: diffuse large B cell lymphoma; R-CHOP: rituximab, cyclophosphamide, doxorubicin: vincristine, and prednisone; NSCLC: non-small cell lung cancer; ICIs: immune checkpoint inhibitor; OS: overall survival; and PFS: progression-free survival

Article Snippet: Rabbit anti-human FN1 IgG antibody , #26836S , Cell Signaling Technology.

Techniques: Comparison

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Spatial transcriptomics reveals prognostically LYZ + fibroblasts and colocalization with FN1 + macrophages in diffuse large B-cell lymphoma

doi: 10.1007/s00262-025-03968-7

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Rabbit anti-human FN1 IgG antibody , #26836S , Cell Signaling Technology.

Techniques: Formalin-fixed Paraffin-Embedded, Gene Expression, RNA Sequencing, Sequencing, Biomarker Discovery, Clinical Proteomics, Microarray, Control, Immunohistochemistry, Microscopy, Immunofluorescence, Software