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: eLife

Article Title: Proteomic landscape of tunneling nanotubes reveals CD9 and CD81 tetraspanins as key regulators

doi: 10.7554/eLife.99172

Figure Lengend Snippet:

Article Snippet: Antibody , Mouse monoclonal anti-ADAM10 11G2 , , Diaclone: #857.800.000 , WB (1/1000).

Techniques: Transfection, Construct, Expressing, Plasmid Preparation, Marker, Sequencing, Purification, Transduction, Control, Software, Staining

Journal: Oncotarget

Article Title: Serological immune response against ADAM10 pro-domain is associated with favourable prognosis in stage III colorectal cancer patients

doi: 10.18632/oncotarget.11181

Figure Lengend Snippet: A. Serological reactivity of Crc patient pool of sera and control pool of sera against the biotinylated protein spot (Av-HRP) that, from the preparative 2DE gel (Coomassie blue), yielded the MS identification of ADAM10; the identity was confirmed by reactivity of an anti-ADAM10 Ab with the same spot. B. Surface expression of ADAM10 in the LS180 Crc cell line. Anti-ADAM10 immunofluorescence reactivity is present on both permeabilized and non-permeabilized cells. Anti-HLA-class I and anti- ß-actin reactivities were used as controls for surface and intracellular expressed proteins, respectively. C. Reactivity of Crc patients and control subjects (Cn) sera against purified ADAM10; anti-ADAM10 Ab reactivity was used for signal normalization. D. - E. Quantitative analysis of serological reactivity reported as normalized OD (mean +/− SEM of 3 experiments in duplicate). D. Testing cohorts Crc1, n = 57; Cn1, n = 39; Crc1-stage I n = 8, stage II n = 17, stage III n = 26, stage IV n = 6. E. Validation cohorts Crc2, n = 49; Cn2, n = 52; Crc2-stage I n = 13, stage II n = 13, stage III n = 13; stage IV n = 10. Statistical analysis was performed by either student-t (S-t) test or Mann-Whitney (M-W) test and non parametric analysis of variance by Kruskal-Wallis (K-W). (*** = p < 0.0001; ** = p < 0.01).

Article Snippet: Human-recombinant ADAM10 (aa 214-672, lacking the pro-domain, R&D Systems 936-AD-020), mouse-recombinant ADAM10 (aa 19-673, including the pro-domain, Calbiochem PF124), ADAM10 pro-domain synthetic peptide (Abcam ab41165; 32 aa within the pro-domain, sequence not available because of property used as immunogen to generate the anti-ADAM10 pro-domain antibody), human-recombinant ADAM17 (aa 1-671, including the pro-domain, Calbiochem PF133) and human ceruloplasmin purified from plasma (Alexis Biochemicals, ALX-200-089) were spotted (200 ng each spot) onto nitrocellulose membrane and tested with the fraction of IgG purified from the sera of six Crc patients considered positive for the presence of auto-antibodies anti-ADAM10 and six patients considered negative.

Techniques: Control, Expressing, Immunofluorescence, Purification, Biomarker Discovery, MANN-WHITNEY

Journal: Oncotarget

Article Title: Serological immune response against ADAM10 pro-domain is associated with favourable prognosis in stage III colorectal cancer patients

doi: 10.18632/oncotarget.11181

Figure Lengend Snippet: A. - D. Learning of the cut-off values for sera categorization (anti-ADAM10 Ab-positive or -negative) performed by ROC analysis of anti-ADAM10 optical density in Crc and Cn. In order to avoid over-fitting, the learning of the cut-off value (that was be applied to the group of patients in a specific stage of the disease) was done using all Crc patients excluding the group of patients at the stage that have been subsequently tested for the follow-up (Crc out Stage … = Crc without patients at specific stage). E. - H. Kaplan-Meier survival curve and Log-rank test analysis in the Crc patients at different stages. The analysis compares the patients showing immunoreactivity against ADAM10 (Ab-pos, solid line) vs. the patients with no reactivity (Ab-neg, dotted line). The considered outcome was recurrence-free survival (RFS) (patient alive, no relapse, no appearance of new metastasis), and the marked “events” were tumor relapse, metastasis and patient death. The dotted red line in panel G indicates the median RFS time significantly changing (from 23 to 55 months) in patients at stage III that showed immunoreactivity against ADAM10.

Article Snippet: Human-recombinant ADAM10 (aa 214-672, lacking the pro-domain, R&D Systems 936-AD-020), mouse-recombinant ADAM10 (aa 19-673, including the pro-domain, Calbiochem PF124), ADAM10 pro-domain synthetic peptide (Abcam ab41165; 32 aa within the pro-domain, sequence not available because of property used as immunogen to generate the anti-ADAM10 pro-domain antibody), human-recombinant ADAM17 (aa 1-671, including the pro-domain, Calbiochem PF133) and human ceruloplasmin purified from plasma (Alexis Biochemicals, ALX-200-089) were spotted (200 ng each spot) onto nitrocellulose membrane and tested with the fraction of IgG purified from the sera of six Crc patients considered positive for the presence of auto-antibodies anti-ADAM10 and six patients considered negative.

Techniques:

Journal: Oncotarget

Article Title: Serological immune response against ADAM10 pro-domain is associated with favourable prognosis in stage III colorectal cancer patients

doi: 10.18632/oncotarget.11181

Figure Lengend Snippet: Cox regression analysis of outcome predicting factor ^°

Article Snippet: Human-recombinant ADAM10 (aa 214-672, lacking the pro-domain, R&D Systems 936-AD-020), mouse-recombinant ADAM10 (aa 19-673, including the pro-domain, Calbiochem PF124), ADAM10 pro-domain synthetic peptide (Abcam ab41165; 32 aa within the pro-domain, sequence not available because of property used as immunogen to generate the anti-ADAM10 pro-domain antibody), human-recombinant ADAM17 (aa 1-671, including the pro-domain, Calbiochem PF133) and human ceruloplasmin purified from plasma (Alexis Biochemicals, ALX-200-089) were spotted (200 ng each spot) onto nitrocellulose membrane and tested with the fraction of IgG purified from the sera of six Crc patients considered positive for the presence of auto-antibodies anti-ADAM10 and six patients considered negative.

Techniques:

Journal: Oncotarget

Article Title: Serological immune response against ADAM10 pro-domain is associated with favourable prognosis in stage III colorectal cancer patients

doi: 10.18632/oncotarget.11181

Figure Lengend Snippet: Immunohistochemistry analysis of ADAM10 expression in normal intestinal epithelia tissue a. and tumoral tissue b. - f. . Arrows indicated in panel a. the few ADAM10 positive monocytes resident in the cryptae, and in panels a. - f. the stronger reactivity of tumor cells invading the stroma.

Article Snippet: Human-recombinant ADAM10 (aa 214-672, lacking the pro-domain, R&D Systems 936-AD-020), mouse-recombinant ADAM10 (aa 19-673, including the pro-domain, Calbiochem PF124), ADAM10 pro-domain synthetic peptide (Abcam ab41165; 32 aa within the pro-domain, sequence not available because of property used as immunogen to generate the anti-ADAM10 pro-domain antibody), human-recombinant ADAM17 (aa 1-671, including the pro-domain, Calbiochem PF133) and human ceruloplasmin purified from plasma (Alexis Biochemicals, ALX-200-089) were spotted (200 ng each spot) onto nitrocellulose membrane and tested with the fraction of IgG purified from the sera of six Crc patients considered positive for the presence of auto-antibodies anti-ADAM10 and six patients considered negative.

Techniques: Immunohistochemistry, Expressing

Journal: Oncotarget

Article Title: Serological immune response against ADAM10 pro-domain is associated with favourable prognosis in stage III colorectal cancer patients

doi: 10.18632/oncotarget.11181

Figure Lengend Snippet: A. Anti-ADAM10 WB analysis in Crc specimens from representative patients with (Ab-pos, C27, C38, C9, C21, C36) or without (Ab-neg, C32, C52) serological reactivity against ADAM10. Anti-β-actin reactivity was used for protein-loading control and OD normalization. B. Quantitative analysis of WB reactivity detected on Ab-pos ( n = 15) and Ab-neg ( n = 12) patient specimens (mean +/− SEM 3 experiments; student's t -test; ** p < 0.01; *** p < 0.001). The ratio between pro-protein and mature ADAM10 signals is reported.

Article Snippet: Human-recombinant ADAM10 (aa 214-672, lacking the pro-domain, R&D Systems 936-AD-020), mouse-recombinant ADAM10 (aa 19-673, including the pro-domain, Calbiochem PF124), ADAM10 pro-domain synthetic peptide (Abcam ab41165; 32 aa within the pro-domain, sequence not available because of property used as immunogen to generate the anti-ADAM10 pro-domain antibody), human-recombinant ADAM17 (aa 1-671, including the pro-domain, Calbiochem PF133) and human ceruloplasmin purified from plasma (Alexis Biochemicals, ALX-200-089) were spotted (200 ng each spot) onto nitrocellulose membrane and tested with the fraction of IgG purified from the sera of six Crc patients considered positive for the presence of auto-antibodies anti-ADAM10 and six patients considered negative.

Techniques: Control

Journal: Oncotarget

Article Title: Serological immune response against ADAM10 pro-domain is associated with favourable prognosis in stage III colorectal cancer patients

doi: 10.18632/oncotarget.11181

Figure Lengend Snippet: ADAM10 pro-domain: reactivity of the rabbit polyclonal anti-ADAM10 pro-domain-specific Ab (ab39178, Abcam), which recognized the immature non-functional isoform of ADAM10, showed patched reactivity. ADAM10 ectodomain: reactivity of the goat polyclonal anti-ADAM10 ectodomain-specific Ab (R&D-Systems, AB936), which recognized both the immature and mature (cleaved and functional) isoforms of ADAM10, showed patched and diffuse signals. Anti-ADAM10 auto-Ab positive serum: reactivity of the human IgG fraction purified from a representative serum (C2) of Crc patients considered positive for the presence of auto-Ab anti ADAM10, showed patched reactivity. Anti-ADAM10 auto-Ab negative serum: no reactivity was observed for the human IgG fraction purified from serum (C25) of a representative Crc patient considered negative for the presence of auto-Ab anti ADAM10. Cell nuclei were stained with Hoechst-33342. The reactivity of the secondary antibodies (goat anti-rabbit IgG Alexa-488; donkey anti-goat IgG Alexa-488 and goat anti-human IgG FITC) are shown as negative controls in . Images were acquired by immunofluorescence microscopy (Zeiss Upright Axo Imager 2 equipped with AxoVision Rel.4.8.2 software); magnification 63X. Images were linearly adjusted for brightness and contrast using Adobe-Photoshop CS4 v.11 software.

Article Snippet: Human-recombinant ADAM10 (aa 214-672, lacking the pro-domain, R&D Systems 936-AD-020), mouse-recombinant ADAM10 (aa 19-673, including the pro-domain, Calbiochem PF124), ADAM10 pro-domain synthetic peptide (Abcam ab41165; 32 aa within the pro-domain, sequence not available because of property used as immunogen to generate the anti-ADAM10 pro-domain antibody), human-recombinant ADAM17 (aa 1-671, including the pro-domain, Calbiochem PF133) and human ceruloplasmin purified from plasma (Alexis Biochemicals, ALX-200-089) were spotted (200 ng each spot) onto nitrocellulose membrane and tested with the fraction of IgG purified from the sera of six Crc patients considered positive for the presence of auto-antibodies anti-ADAM10 and six patients considered negative.

Techniques: Functional Assay, Purification, Staining, Immunofluorescence, Microscopy, Software

Journal: Oncotarget

Article Title: Serological immune response against ADAM10 pro-domain is associated with favourable prognosis in stage III colorectal cancer patients

doi: 10.18632/oncotarget.11181

Figure Lengend Snippet: Double staining was performed with rabbit anti-ADAM10 pro-domain (ab39178, Abcam), and either IgGs purified from Crc patient serum or mouse anti-HLA class I (Santa-Cruz Biotechnology). Secondary Abs were Alexa-546-conjugated goat-anti-rabbit IgG and Alexa-488-conjugated rabbit-anti-mouse IgG or FITC-conjugated goat-anti-human IgG. Cell nuclei were stained with Hoechst-33342, and differential interference contrast (DIC) images were also acquired. Staining was assessed by immunofluorescence confocal microscopy (Leica TCS SP5 Laser Scanning Confocal) and images acquired with LAS-AF (Leica) software; magnification 63X. Images from single channel and merged images are shown. Images were linearly adjusted for brightness and contrast using ImageJ 1.47v software.

Article Snippet: Human-recombinant ADAM10 (aa 214-672, lacking the pro-domain, R&D Systems 936-AD-020), mouse-recombinant ADAM10 (aa 19-673, including the pro-domain, Calbiochem PF124), ADAM10 pro-domain synthetic peptide (Abcam ab41165; 32 aa within the pro-domain, sequence not available because of property used as immunogen to generate the anti-ADAM10 pro-domain antibody), human-recombinant ADAM17 (aa 1-671, including the pro-domain, Calbiochem PF133) and human ceruloplasmin purified from plasma (Alexis Biochemicals, ALX-200-089) were spotted (200 ng each spot) onto nitrocellulose membrane and tested with the fraction of IgG purified from the sera of six Crc patients considered positive for the presence of auto-antibodies anti-ADAM10 and six patients considered negative.

Techniques: Double Staining, Purification, Staining, Immunofluorescence, Confocal Microscopy, Software

Journal: Oncotarget

Article Title: Serological immune response against ADAM10 pro-domain is associated with favourable prognosis in stage III colorectal cancer patients

doi: 10.18632/oncotarget.11181

Figure Lengend Snippet: Anti-ADAM10 pro-domain: reactivity of the rabbit anti-ADAM10 pro-domain Ab (ab39178, Abcam) after competition with control rabbit IgG. Anti-ADAM10 positive serum (C2): reactivity of the IgG fraction purified from a representative serum of a Crc patient considered positive for the presence of auto-Abs anti ADAM10 after competition either with control rabbit IgGs or the anti-ADAM10 pro-domain Ab. Anti-ADAM10 negative serum (C32): reactivity of the IgG fraction purified from a representative serum of a Crc patient considered negative for the presence of anti ADAM10 auto-Ab after competition either with control rabbit IgGs or anti-ADAM10 pro-domain Ab. Cell nuclei were stained with Hoechst-33342; secondary Abs were goat anti-rabbit IgG Alexa-488 and goat anti-human IgG FITC. Images were acquired by immunofluorescence microscopy (Zeiss Upright Axo Imager 2 equipped with AxoVision Rel.4.8.2 software); magnification 63X. Images were linearly adjusted for brightness and contrast using Adobe-Photoshop CS4 v.11 software.

Article Snippet: Human-recombinant ADAM10 (aa 214-672, lacking the pro-domain, R&D Systems 936-AD-020), mouse-recombinant ADAM10 (aa 19-673, including the pro-domain, Calbiochem PF124), ADAM10 pro-domain synthetic peptide (Abcam ab41165; 32 aa within the pro-domain, sequence not available because of property used as immunogen to generate the anti-ADAM10 pro-domain antibody), human-recombinant ADAM17 (aa 1-671, including the pro-domain, Calbiochem PF133) and human ceruloplasmin purified from plasma (Alexis Biochemicals, ALX-200-089) were spotted (200 ng each spot) onto nitrocellulose membrane and tested with the fraction of IgG purified from the sera of six Crc patients considered positive for the presence of auto-antibodies anti-ADAM10 and six patients considered negative.

Techniques: Control, Purification, Staining, Immunofluorescence, Microscopy, Software

Journal: Oncotarget

Article Title: Serological immune response against ADAM10 pro-domain is associated with favourable prognosis in stage III colorectal cancer patients

doi: 10.18632/oncotarget.11181

Figure Lengend Snippet: The reactivity of the IgG fraction purified from the sera of 6 Crc patients positive for the presence of auto-Abs anti-ADAM10 (C2, C27, C20, C9, C14, C19) and 6 Crc patients considered negative (C32, C50, C25, C11, C13, C45) was tested on human-recombinant ADAM10 (mature form lacking the pro-domain), mouse-recombinant ADAM10 (immature form including the pro-domain), ADAM10 pro-domain synthetic peptide (32 aa within the pro-domain sequence), human-recombinant ADAM17 (immature form including the pro-domain) and purified human ceruloplasmin (Cp) proteins spotted (200 ng/spot) onto nitrocellulose membrane. The reactivity of the anti-ADAM10 ectodomain Ab that recognizes both mature and immature ADAM10, anti-ADAM10 pro-domain Ab, anti-ADAM17 and anti-ceruloplasmin Abs were used as controls.

Article Snippet: Human-recombinant ADAM10 (aa 214-672, lacking the pro-domain, R&D Systems 936-AD-020), mouse-recombinant ADAM10 (aa 19-673, including the pro-domain, Calbiochem PF124), ADAM10 pro-domain synthetic peptide (Abcam ab41165; 32 aa within the pro-domain, sequence not available because of property used as immunogen to generate the anti-ADAM10 pro-domain antibody), human-recombinant ADAM17 (aa 1-671, including the pro-domain, Calbiochem PF133) and human ceruloplasmin purified from plasma (Alexis Biochemicals, ALX-200-089) were spotted (200 ng each spot) onto nitrocellulose membrane and tested with the fraction of IgG purified from the sera of six Crc patients considered positive for the presence of auto-antibodies anti-ADAM10 and six patients considered negative.

Techniques: Purification, Recombinant, Sequencing, Membrane

Journal: Kidney international

Article Title: Characterization of CXCL16 and ADAM10 in the normal and transplanted kidney.

doi: 10.1038/ki.2008.181

Figure Lengend Snippet: Figure 2 | Influence of metalloproteinases on the expression and release of CXCL16. (a) Analysis of soluble CXCL16 released from primary TALDCs with a CXCL16-specific ELISA. Cells were preincubated with different proteinase inhibitors for 15 min before IFN-g (24 ng/ml) was added for 24 h. Application of IFN-g (24 ng/ml) increased the amount of soluble CXCL16 in the supernatants of TALDCs. The ADAM10-specific metalloproteinase inhibitor, GI254023X (GI), and the broad-spectrum metalloproteinase inhibitors, GM6001 and TAPI-2, reduced the IFN-g-induced CXCL16 release. Data are mean±s.d. (n ¼ 3); ***Po0.001 versus control; ###Po0.001, ##Po0.01; #Po0.05 versus cytokine-treated cells. (b) Inhibition of metalloproteinases increased cellular CXCL16 protein expression in TALDCs. Fifteen minutes before application of IFN-g, cells were pretreated with the indicated concentration of the metalloproteinase inhibitors GI254023X, GM6001, and TAPI-2. CXCL16 protein levels were measured by western blot analysis and b-actin was used as a loading control. (c) Knockdown of CXCL16 with CXCL16-specific siRNA in TALDCs is shown by western blot analysis. (d) Application of pharmacological inhibitors of metalloproteinases increased the amount of cellular CXCL16. Cell lysates were isolated as described under Materials and Methods, and 15 mg total proteins were used to measure the amount of cell-expressed CXCL16 by CXCL16-specific ELISA. Data are mean þ s.d. (n ¼ 5). Statistically significant release of CXCL16 (Po0.001) is indicated by asterisk. (e) Suppression of ADAM10 protein levels by RNA interference in the presence and absence of IFN-g. Cell lysates of TALDCs were prepared 48 h after transfection with siRNA specific for ADAM10 and an unspecific siRNA. Mock-transfected cells treated with the transfection lipid in the absence of siRNA were used as a control. Western blot analysis of ADAM10 protein expression was performed. Blots were reprobed with an antibody specific for b-actin as a loading control. (f) Inhibition of ADAM10 by siRNA reduced the IFN-g-stimulated CXCL16 release in TALDCs. Supernatants of siRNA-treated and IFN-g- stimulated or unstimulated cells were analyzed for soluble CXCL16 using a CXCL16-specific ELISA (n ¼ 3); ***Po0.001 versus control; ###Po0.001 versus IFNg-treated cells.

Article Snippet: Recombinant human CXCL16, recombinant human IFN-g, anti-human ADAM10 ectodomain antibody, and anti-human CXCR6 antibody were obtained from R&D Systems (Wiesbaden, Germany).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Control, Inhibition, Concentration Assay, Western Blot, Knockdown, Isolation, Transfection

Journal: Kidney international

Article Title: Characterization of CXCL16 and ADAM10 in the normal and transplanted kidney.

doi: 10.1038/ki.2008.181

Figure Lengend Snippet: Figure 3 | Localization of ADAM10 protein in normal human kidney. (a) In the overview, ADAM10 protein is mainly expressed in tubular cells, but expression was also found in glomerular cells. Serial sections of normal renal tissue represented constitutive ADAM10 expression in aquaporin-2 (e), calbindin D-28K (f), and Tamm–Horsfall glycoprotein (g) expressing tubular profiles, which correspond to CD, (DCT, CNT), and TAL, respectively. Proximal tubules were devoid of any specific ADAM10 staining. (h) Immunofluorescent detection of ADAM10 (red fluorescence) in aquaporin-2-positive principal cells of the CD (green fluorescence). A prominent overlap of both signals in the same cells can be seen, indicated in the examples by asterisks. In contrast, arrows indicate cells negative for either aquaporin-2 or ADAM10, thus representing presumable intercalated cells. (i) ADAM10 is not expressed in the intercalated cells of the CD. Note: arrows represent H þ-ATPase-expressing intercalated cells (green colour) that do not express ADAM10 (red colour); compare with the merged view. (j) Confocal immunofluorescence analysis of ADAM10 and CXCL16 expression in a cortical CD. Tissue section was stained with Alexa Fluor 488 and Cy3 secondary antibodies to visualize the localization of CXCL16 (red) and ADAM10 (green) proteins, respectively. Both signals can be confined to same segment-specific cells; see merged picture on the right.

Article Snippet: Recombinant human CXCL16, recombinant human IFN-g, anti-human ADAM10 ectodomain antibody, and anti-human CXCR6 antibody were obtained from R&D Systems (Wiesbaden, Germany).

Techniques: Expressing, Staining, Fluorescence, Immunofluorescence

Journal: Kidney international

Article Title: Characterization of CXCL16 and ADAM10 in the normal and transplanted kidney.

doi: 10.1038/ki.2008.181

Figure Lengend Snippet: Figure 4 | Supernatants from ADAM10- and CXCL16 siRNA-transfected TALDCs decreased the chemotaxis of Jurkat T cells. (a) Surface expression of the chemokine receptor CXCR6 in Jurkat T cells analyzed by fluorescence-activated cell sorting analysis. (b) Inhibition of CXCL16 protein expression by siRNA fully abolished release of CXCL16 in TALDCs. Cells were transfected with different siRNA duplexes for inhibition of ADAM10 or CXCL16, an unspecific siRNA (scrambled), or not transfected with siRNA (mock). A total of 24 h after transfection, TALDCs were stimulated with IFN-g for 24 h, where indicated, and supernatants were collected. Soluble CXCL16 was determined by a CXCL16-specific ELISA (n ¼ 4). Data are mean±s.d.; ***Po0.001 versus control; ###Po0.001 versus IFN-g-treated cells. (c) Decrease of soluble CXCL16 correlates with a reduction in the migration of Jurkat T cells. Supernatants shown in Figure 5b were used for chemotaxis assays (n ¼ 2). Recombinant CXCL16 induced migration of CXCR6-expressing Jurkat T cells about threefold; **Po0.01; *Po0.05 versus control.

Article Snippet: Recombinant human CXCL16, recombinant human IFN-g, anti-human ADAM10 ectodomain antibody, and anti-human CXCR6 antibody were obtained from R&D Systems (Wiesbaden, Germany).

Techniques: Transfection, Chemotaxis Assay, Expressing, Fluorescence, FACS, Inhibition, Enzyme-linked Immunosorbent Assay, Control, Migration, Recombinant

Journal: Kidney international

Article Title: Characterization of CXCL16 and ADAM10 in the normal and transplanted kidney.

doi: 10.1038/ki.2008.181

Figure Lengend Snippet: Figure 5 | Increased urinary CXCL16 correlated with focally apical CXCL16 expression in tubular cells of renal allografts with the histopathological diagnosis of ATN. Six allograft biopsies of patients diagnosed with ATN (cases A1–A6; clinical parameters and CXCL16 analysis are shown in Table 1) were analyzed by CXCL16 immunohistochemical analysis (a–f) and with ADAM10 (green colour) and CXCL16 (red colour) double immunofluorescence staining (g–l). Notably, all patients showed focally increased apical CXCL16 expression in renal tubuli. Strongest apical CXCL16, seen in patient A4 (d), correlated with the highest amount of urinary CXCL16 (Table 1). In contrast, ADAM10 expression (green colour) was not significantly changed in ATN patients (A1–A6) compared with normal kidney (g–l). (m) Urinary CXCL16 measured by a CXCL16-specific ELISA in healthy volunteers (normal), in patients with IR, and in patients with ATN. Data are mean±s.d.; **Po0.01 versus control.

Article Snippet: Recombinant human CXCL16, recombinant human IFN-g, anti-human ADAM10 ectodomain antibody, and anti-human CXCR6 antibody were obtained from R&D Systems (Wiesbaden, Germany).

Techniques: Expressing, Biomarker Discovery, Immunohistochemical staining, Double Immunofluorescence Staining, Enzyme-linked Immunosorbent Assay, Control

Journal: Kidney international

Article Title: Characterization of CXCL16 and ADAM10 in the normal and transplanted kidney.

doi: 10.1038/ki.2008.181

Figure Lengend Snippet: Figure 6 | Increased ADAM10 expression in allograft biopsies of kidney transplant patient with the clinical and histopathological diagnosis of acute IR. Renal allograft biopsies of patients diagnosed with IR were analyzed by double immunofluorescence for ADAM10 and CXCL16 expression in comparison with normal kidney (upper panel). Clinical features and ADAM10 analysis of patients are listed in Table 2. Tissue sections were stained with Alexa Fluor 488 and Cy3 secondary antibodies to visualize the localization of CXCL16 (red) and ADAM10 (green) proteins, respectively. In some tubuli of patient B1 (marked with arrows), strong basolateral ADAM10 expression and no CXCL16 expression could be seen (middle panel). In contrast, in few tubuli of the same patient (marked with a star) both molecules colocalized. In the lower panel (patient B5), strong coexpression of ADAM10 and CXCL16 in a tubule is visible, accompanied with interstitial inflammatory infiltrates.

Article Snippet: Recombinant human CXCL16, recombinant human IFN-g, anti-human ADAM10 ectodomain antibody, and anti-human CXCR6 antibody were obtained from R&D Systems (Wiesbaden, Germany).

Techniques: Expressing, Biomarker Discovery, Immunofluorescence, Comparison, Staining