Journal: bioRxiv

Article Title: Development of Anti-Inflammatory Extracellular Vesicles by Surface Expression of Syndecan-4

doi: 10.1101/2025.07.05.663259

Figure Lengend Snippet: a) The morphology of L-EVs, S-EVs, and miniEVs from HEK293F after ultracentrifugation (called “isolation” – upper panels) and after iodixanol density gradient (called “purification” – lower panels), visualized by negative-stained transmission electron microscopy (TEM). b) Diameter of HEK293F EVs measured manually on TEM images. c-e) The purity (measured as particles per protein) of EVs after different ultracentrifugation (called “isolation”) and after iodixanol density gradient (called “purification”). f) Western blot for CD9, CD81, CD63, ADAM10, Flotillin-1 and Calnexin in HEK293F EVs. g) Surface expression of CD9, CD81 and CD63 in HEK293F EVs measured by Nano-FCM. Data were analyzed using one-way ANOVA and student t test . *, P <0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Abbreviations: EVs, extracellular vesicles; L-EVs, large EVs; S-EVs, small EVs.

Article Snippet: The primary antibodies were as follows: CD9 (EMD Millipore, 1:1000), CD81 (Abcam, 1:1000), CD63 (BD Pharmingen, 1:1000), ADAM10 (Rnd Systems, 1:500), Flotilin-1 (Abcam, 1:1000), Calnexin (Cell Signalling Technology, 1:1000), Heparan Sulfate (Sigma Aldrich, 1:1000), β-actin (Cell Signalling Technology, 1:1000).

Techniques: Isolation, Purification, Staining, Transmission Assay, Electron Microscopy, Western Blot, Expressing

Journal: PLoS Pathogens

Article Title: Pseudomonas aeruginosa ExlA and Serratia marcescens ShlA trigger cadherin cleavage by promoting calcium influx and ADAM10 activation

doi: 10.1371/journal.ppat.1006579

Figure Lengend Snippet: A . A549 cells were either treated with 0.1 μg/mL PMA or 5 μM ionomycin (Iono) for 30 min, or infected with CLJ1 (90 min), or left untreated/uninfected (NI). Cellular extracts were analysed by Western blot using E-cadherin and β-actin antibodies. FL, full-length; CTF, C-terminal fragment. The experiment was performed twice. B . A549 cells (left) or HUVECs (right) were pre-treated with DMSO, the general metalloprotease inhibitor GM6001 (10 μg/mL) or the specific ADAM10 inhibitor GI254023X (5 μM) and then incubated with CLJ1 or IHMA87 (90 min), or uninfected (NI). Cellular extracts were analysed as above. The experiment was performed twice for A549 and 3 times for HUVECs. C . A549 or ADAM10-deficient A549 (A549 ADAM10 -/- ) cells were incubated with either CLJ1 or IHMA87. Cellular extracts were prepared at different time points post-infection as indicated and analysed by Western blot (left). The right panel shows the FACS analysis of ADAM10 surface expression of both cell lines, as well as the negative control. The experiment was performed 3 times. D . Similar experiment with HUVECs, either transfected with ADAM10 siRNA or untreated. The experiment was performed twice.

Article Snippet: Three days after transfection, cells were analysed by FACScalibur flow cytometer (Becton Dickinson) after staining with ADAM10 antibody (R&D Systems) and anti-mouse-Alexa 488 antibody (Molecular Probes).

Techniques: Infection, Western Blot, Incubation, Expressing, Negative Control, Transfection

Journal: PLoS Pathogens

Article Title: Pseudomonas aeruginosa ExlA and Serratia marcescens ShlA trigger cadherin cleavage by promoting calcium influx and ADAM10 activation

doi: 10.1371/journal.ppat.1006579

Figure Lengend Snippet: Plasma membrane rupture was monitored by LDH release in the supernatant. A549 or A549 ADAM10 -/- cells were incubated for 5 hours with IHMA87, IHMA87Δ exlA or IHMA87Δ exlA/exlA strains. The supernatants were the tested for LDH activity. The histograms show the mean ± s.d. of triplicates. The data are representative of 3 experiments.

Article Snippet: Three days after transfection, cells were analysed by FACScalibur flow cytometer (Becton Dickinson) after staining with ADAM10 antibody (R&D Systems) and anti-mouse-Alexa 488 antibody (Molecular Probes).

Techniques: Clinical Proteomics, Membrane, Incubation, Activity Assay

Journal: PLoS Pathogens

Article Title: Pseudomonas aeruginosa ExlA and Serratia marcescens ShlA trigger cadherin cleavage by promoting calcium influx and ADAM10 activation

doi: 10.1371/journal.ppat.1006579

Figure Lengend Snippet: A . A549 cells were incubated with various concentrations of TFP, as indicated, to impede calmodulin interaction with ADAM10. Ionomycin was used as positive controls. E-cadherin cleavage was assessed by Western blot. The experiment was performed twice. B . Western blot analysis of A549 E-cadherin contents after infection with CLJ1 or IHMA87, in presence or absence of BAPTA-AM. Both experiments were performed 3 times. C . LDH release of A549 cells infected with either CLJ1 or IHMA87, in presence/ absence of BAPTA-AM. Student’s t-test showed significance between the two treatments for both CLJ1 and IHMA87 data (p-values indicated above the bars). The experiment was performed 3 times.

Article Snippet: Three days after transfection, cells were analysed by FACScalibur flow cytometer (Becton Dickinson) after staining with ADAM10 antibody (R&D Systems) and anti-mouse-Alexa 488 antibody (Molecular Probes).

Techniques: Incubation, Western Blot, Infection

Journal: PLoS Pathogens

Article Title: Pseudomonas aeruginosa ExlA and Serratia marcescens ShlA trigger cadherin cleavage by promoting calcium influx and ADAM10 activation

doi: 10.1371/journal.ppat.1006579

Figure Lengend Snippet: A . A549 cells (left) or HUVECs (right) were incubated with the S . marcescens ShlA-secreting strain Db11, or with the non-ShlA-secreting mutant 21C4. Cellular extracts were analysed for their E- or VE-cadherin contents. The experiment was performed twice for the left panel and once for the right panel. B . Similar analysis using A549 ADAM10 -/- . The experiment was performed once. C . Similar analysis using A549 cells, in presence/ absence of BAPTA-AM. D-G . Intracellular Ca 2+ contents and plasma membrane permeability were measured using Fluo3-AM and Draq7 fluorescent probes, respectively. A549 cells ( D,F ) and HUVECs ( E,G ) were infected with Db11 ( D,E ) or 21C4 ( F,G ) and fluorescence was recorded on both channels by videomicroscopy. Five cells were analysed in each case; the Fluo3 intensities are represented by straight lines and the Draq7 intensities by dashed lines, using the same colour code for one cell. Data are representative of 8 and 5 independent experiments for A549 and HUVECs, respectively.

Article Snippet: Three days after transfection, cells were analysed by FACScalibur flow cytometer (Becton Dickinson) after staining with ADAM10 antibody (R&D Systems) and anti-mouse-Alexa 488 antibody (Molecular Probes).

Techniques: Incubation, Mutagenesis, Clinical Proteomics, Membrane, Permeability, Infection, Fluorescence

Journal: PLoS Pathogens

Article Title: Pseudomonas aeruginosa ExlA and Serratia marcescens ShlA trigger cadherin cleavage by promoting calcium influx and ADAM10 activation

doi: 10.1371/journal.ppat.1006579

Figure Lengend Snippet: In uninfected cells, pro-ADAM10 is associated with calmodulin, preventing its maturation and export to the plasma membrane. Pore formation by ExlA or ShlA induces a massive Ca 2+ influx in host cells. Intracellular Ca 2+ interacts with the Ca 2+ -binding protein calmodulin, which detaches from pro-ADAM10, allowing its maturation to m-ADAM10. m-ADAM10 cleaves E- and VE-cadherin in epithelial and endothelial cells, respectively, provoking intercellular junction rupture.

Article Snippet: Three days after transfection, cells were analysed by FACScalibur flow cytometer (Becton Dickinson) after staining with ADAM10 antibody (R&D Systems) and anti-mouse-Alexa 488 antibody (Molecular Probes).

Techniques: Clinical Proteomics, Membrane, Binding Assay

Journal: Scientific Reports

Article Title: The proteomic analysis of breast cell line exosomes reveals disease patterns and potential biomarkers

doi: 10.1038/s41598-020-70393-4

Figure Lengend Snippet: List of the membrane proteins suggested as potential breast cancer biomarkers.

Article Snippet: For the validation of protein biomarkers, isolated exosomes were incubated for 40 min with one of the following primary antibodies: Glut-1 (Invitrogen, MA5-31960, SA0377), GLYP-1 (Invitrogen, PA5-86043, polyclonal) and ADAM10 (MyBioSource, MBS435195, polyclonal).

Techniques: Membrane

Journal: Scientific Reports

Article Title: The proteomic analysis of breast cell line exosomes reveals disease patterns and potential biomarkers

doi: 10.1038/s41598-020-70393-4

Figure Lengend Snippet: Western blot analysis of CD63, CD81, GPC-1, GLUT-1, and ADAM10 in the exosomes derived from the MDA and MCF cell lines. Full-length gels are shown in Figure .

Article Snippet: For the validation of protein biomarkers, isolated exosomes were incubated for 40 min with one of the following primary antibodies: Glut-1 (Invitrogen, MA5-31960, SA0377), GLYP-1 (Invitrogen, PA5-86043, polyclonal) and ADAM10 (MyBioSource, MBS435195, polyclonal).

Techniques: Western Blot, Derivative Assay

Journal: Scientific Reports

Article Title: The proteomic analysis of breast cell line exosomes reveals disease patterns and potential biomarkers

doi: 10.1038/s41598-020-70393-4

Figure Lengend Snippet: tSNE plots of exosomes analyzed by FACS showing expression levels of ( a ) GPC-1 ( b ) GLUT-1 and ( c ) ADAM10 along with CD81 and CD63 on the surface of BC-derived exosomes.

Article Snippet: For the validation of protein biomarkers, isolated exosomes were incubated for 40 min with one of the following primary antibodies: Glut-1 (Invitrogen, MA5-31960, SA0377), GLYP-1 (Invitrogen, PA5-86043, polyclonal) and ADAM10 (MyBioSource, MBS435195, polyclonal).

Techniques: Expressing, Derivative Assay