Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Cleavage of annexin A1 by ADAM10 during secondary necrosis generates a monocytic "find-me" signal.

doi: 10.4049/jimmunol.1004073

Figure Lengend Snippet: FIGURE 3. Anx A1 cleavage is downstream of ADAM10. A, Anx A1 cleavage cannot be blocked by inhibition of elastase or proteinase 3. Jurkat cells were stimulated with 2.5 mM staurosporine in the absence or presence of elastase inhibitor (Ela-I), aprotinin, or Pefabloc for 18 h. Subsequently, cells were lysed, and anx A1 processing was monitored by immunoblot analysis. B, Anx A1 cleavage is mediated by a membrane-resident protease. One microgram of purified recombinant human anx A1 was incubated with the culture supernatant of 4 3 106 secondary necrotic Jurkat cells per milliliter or the membrane fraction of 3 3 107 secondary necrotic Jurkat cells at 37˚C for the indicated times, and anx A1 cleavage was examined by immunoblot analysis. C, Anx A1 cleavage is blocked by o-phenanthroline. Jurkat cells were stimulated as in A in the absence or presence of the metalloproteinase inhibitor o-phenanthroline. Anx A1 processing was detected by immunoblot analysis. Vinculin was used as a loading control. D, Addition of the broad-range matrix metalloproteinase inhibitor GM 6001 blocks anx A1 cleavage. Jurkat cells were stimulated as in A in the presence of 0–100 mM GM 6001. Afterwards, anx A1 cleavage was monitored by immunoblot analysis. PARP was used as a loading and apoptosis/secondary necrosis control. E, Proteolytic processing of anx A1 can be blocked by the ADAM10 inhibitor GI 254023X. Jurkat cells were stimulated as in A in the absence or presence of 10 mM of the ADAM10 inhibitor GI 254023X (GI) or the ADAM10/17 inhibitor GW 280264X (GW). Subsequently, anti-anx A1 immunoblot analysis was performed with protein extracts as in D. F, Analysis of ADAM10 and ADAM17 knockdown efficiency by qRT-PCR. Knockdown of ADAM10 and ADAM17 expression was carried out by electroporation of Jurkat cells with two different ADAM10- and ADAM17-specific oligonucleotides and a scramble control oligonucleotide as described in Materials and Methods. Total RNA was prepared, reversely transcribed, and the resulting cDNA was used for qRT-PCR as described in Materials and Methods. Relative ADAM10/17 mRNA levels were normalized on the endogenous control ALAS-1, and the ADAM10/17 mRNA level in Jurkat cells that were treated with the scramble control siRNA was set as 100% calibrator. G, Anx A1 cleavage is strongly inhibited in ADAM10 silenced cells. siRNA- mediated knockdown of ADAM10 or ADAM17 expression was performed as in F. Subsequently, cells were stimulated to undergo secondary necrosis as in A, and anx A1 cleavage was monitored by immunoblot analysis. The amount of anx A1 p36 compared with total anx A1 was calculated from integrated pixel intensities, and the inhibition of cleavage is presented as percent of the scramble control. PARP served as a loading and apoptosis/secondary necrosis control.

Article Snippet: Recombinant human MCP-1, SDF-1a, ADAM10, and ADAM17 ectodomains were obtained from R&D Systems.

Techniques: Inhibition, Western Blot, Membrane, Recombinant, Incubation, Control, Knockdown, Quantitative RT-PCR, Expressing, Electroporation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Cleavage of annexin A1 by ADAM10 during secondary necrosis generates a monocytic "find-me" signal.

doi: 10.4049/jimmunol.1004073

Figure Lengend Snippet: FIGURE 4. Anx A1 is directly cleaved by ADAM10 after F7. A, Domain structure of different anx A1 constructs. Arabic numbers depict the amino acid position, and annexin repeats are numbered I–IV. B, Recombinant human anx A1 (aa 1–346) is processed by recombinant human ADAM10. One mi- crogram of recombinant human anx A1 (aa 1–346) was incubated with 100 ng recombinant human ADAM10 ectodomain in the presence or absence of 100 mM of the matrix metalloproteinase inhibitor TAPI-2 at 37˚C for the indicated times. Subsequently, anx A1 cleavage was detected by SDS-PAGE and immunoblot analysis with an anti-anx A1 Ab. C, Recombinant human anx A1 core domain (aa 47–346) is not cleaved by ADAM10. Incubation of anx A1 (aa 47–346) with ADAM10 ectodomain was performed as in B. For immunoblot analysis, a polyclonal anti-anx A1 Ab was used. D, The cleavage site of ADAM10 is located within the unique N-terminal domain of anx A1 (aa 1–46). One microgram of the recombinant human anx A1 N-terminal domain (aa 1–46) was incubated with ADAM10 as in B. Cleavage fragments were separated by SDS-PAGE and visualized by subsequent silver staining. E, Recombinant human anx A1 (aa 1–346) is not processed by recombinant human ADAM17. One microgram of recombinant human anx A1 (aa 1–346) was incubated with 100 ng of recombinant human ADAM17 ectodomain as in B. F, Identification of the ADAM10 cleavage site within the anx A1 N-terminal domain. Recombinant human anx A1 (aa 1–346) was incubated with native or heat-inactivated recombinant human ADAM10 as in A. Subsequently, the reaction mixture was subjected to N-terminal Edman degradation. The N-terminal sequence newly generated by incubation with active ADAM10 was L8

Article Snippet: Recombinant human MCP-1, SDF-1a, ADAM10, and ADAM17 ectodomains were obtained from R&D Systems.

Techniques: Construct, Recombinant, Incubation, SDS Page, Western Blot, Silver Staining, Sequencing, Generated

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Cleavage of annexin A1 by ADAM10 during secondary necrosis generates a monocytic "find-me" signal.

doi: 10.4049/jimmunol.1004073

Figure Lengend Snippet: FIGURE 6. ADAM10 contributes to the release of “find-me” signals from secondary necrotic cells. A, GM 6001 inhibits the release of monocytic chemoattractants during secondary necrosis. Jurkat, MOLT-4, and THP-1 cells were UV-irradiated and incubated for 12 or 18 h in the presence or absence of the broad-range matrix metalloproteinase inhibitor GM 6001 (100 mM). Cell-free culture supernatants were collected and analyzed for their chemotactic potential as in Fig. 5A. Error bars represent SD of quadruplicates. B, The release of secondary necrotic cell-derived attraction signals is strongly decreased in the presence of the ADAM10-specific inhibitor GI 254023X. Jurkat cells were UV-irradiated and incubated for 18 h in the absence or presence of 10 mM of the ADAM10 inhibitor GI 254023X (GI) or the ADAM10/17 inhibitor GW 280264X (GW). Supernatants were collected and applied to a transmigration assay with THP-1 cells as in Fig. 5A. Error bars represent SD of quadruplicates. C, Evaluation of ADAM10 knockdown efficiency by FACS analysis. Knockdown of ADAM10 expression in Jurkat cells was carried out as in Fig. 3G. On day 5 after the first electroporation, cells were fixed, permeabilized, and stained with anti-ADAM10–PE Ab to detect ADAM10 expression level by FACS analysis or IgG-2b-PE isotype control. Left panel, Representative histograms are shown. Right panel, Median PE fluorescence of the histograms in the left panel. D, The release of monocytic attraction signals is strongly decreased in ADAM10-silenced secondary necrotic cells. ADAM10-silenced cells were induced to undergo secondary necrosis by UV irradiation. Cell culture supernatants were harvested and analyzed for their chemotactic potential in a transmigration assay with THP-1 cells as in Fig. 5A. Mean values + SD of quadruplicates are given.

Article Snippet: Recombinant human MCP-1, SDF-1a, ADAM10, and ADAM17 ectodomains were obtained from R&D Systems.

Techniques: Irradiation, Incubation, Derivative Assay, Transmigration Assay, Knockdown, Expressing, Electroporation, Staining, Control, Cell Culture

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: The Journal of Biological Chemistry

Article Title: ADAM17 mediates ectodomain shedding of the soluble VLDL receptor fragment in the retinal epithelium

doi: 10.1016/j.jbc.2021.101185

Figure Lengend Snippet: Effect of various proteinase inhibitors on VLDLR shedding. Primary human RPE cells were transduced with adenovirus (MOI = 25) expressing VLDLRII (Ad-VLDLRII) or with Ad-RFP as control for 24 h. Then, culture media were replaced by serum-free DMEM containing various proteinase inhibitors (10 μM) for another 24 h. After that, the culture media and cell lysates were harvested for Western blot analysis. A , representative images of Western blotting for soluble ectodomain of VLDLR (sVLDLR) in culture media and the full-length VLDLR in cell lysates. PMA (a PKC activator) and proteinase inhibitors including TAPI-1 (tumor necrosis factor-α proteinase inhibitor), GM6001 (pan-MMP inhibitor), GI254023x (ADAM10 inhibitor), and GW280264x (ADAM17 inhibitor) were used. The same volume of DMSO (vehicle) was used as control. B , densitometry analysis of sVLDLR in culture media normalized by the full-length VLDLR levels in cell lysates in ( A ) (n = 3). Cells with Ad-VLDLRII transduction and DMSO treatment were used as control. C , representative images of Western blotting for sVLDLR and VLDLR from primary human RPE cells treated with indicated doses of GW280264x or DMSO as control for 24 h. D , densitometry of sVLDLR in culture media normalized by VLDLR in cell lysates in ( C ) (n = 3). E , ADAM17 activity in GW280264x-treated cells were measured using an ADAM17 activity assay kit (n = 3). Data were presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. ADAM17, a disintegrin and metalloprotease 17; DMEM, Dulbecco's modified Eagle's medium; DMSO, dimethyl sulfoxide; MOI, multiplicity of infection; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; RFP, red fluorescent protein; RPE, retinal pigment epithelium; VLDLR, very low-density lipoprotein receptor; VLDLRII, VLDLR variant II.

Article Snippet: Expression vectors for human ADAM10 (Addgene; plasmid #31717) and human ADAM17 (Addgene; plasmid #31713) were gifts from Dr Rik Derynck ( ).

Techniques: Transduction, Expressing, Control, Western Blot, Activity Assay, Modification, Infection, Variant Assay

Journal: The Journal of Biological Chemistry

Article Title: ADAM17 mediates ectodomain shedding of the soluble VLDL receptor fragment in the retinal epithelium

doi: 10.1016/j.jbc.2021.101185

Figure Lengend Snippet: Effect of siRNA knockdown and plasmid overexpression of ADAM10 and ADAM17 on VLDLR shedding. Primary human RPE cells were transiently transfected with indicated siRNA (50 nM) or plasmid (1 μg/ml) for 24 h. Cells were subsequently infected with Ad-VLDLRII (MOI = 25) for 24 h. Then, culture media were replaced with serum-free DMEM for another 24 h. Finally, culture media and cell lysates were collected for Western blot analysis. A , representative images of Western blotting for sVLDLR in culture media and full-length VLDLR, ADAM10, and ADAM17 in cell lysates. The RPE cells were transfected with the siRNA for ADAM10, ADAM17, or control siRNA. B , quantification of densitometry of sVLDLR in culture media normalized by VLDLR in cell lysates in ( A ) (n = 3). C , representative images of Western blotting for sVLDLR in culture media, and VLDLR, ADAM10, and ADAM17 in cells transfected with plasmid overexpressing human ADAM10, human ADAM17, or RFP as control. D , protein levels of sVLDLR in the media in ( C ) were quantified and normalized by the full-length VLDLR in cell lysates (n = 3). E , representative images of Western blotting of sVLDLR in culture media, VLDLR, ADAM10, and ADAM17 in human primary RPE cells, which were transfected with siRNA knocking down human ADAM17 (siADAM17) or plasmid overexpressing human ADAM17 (pADAM17). F , quantification of densitometry of sVLDLR in culture media normalized by VLDLR in cell lysates in ( E ) (n = 3). In A , C , and E , p and m indicated precursor and mature forms of ADAM10 and ADAM17, respectively. Data were presented as mean ± SD. ∗∗ p < 0.01. ADAM10, a disintegrin and metalloprotease 10; ADAM17, a disintegrin and metalloprotease 17; DMEM, Dulbecco's modified Eagle's medium; MOI, multiplicity of infection; RFP, red fluorescent protein; RPE, retinal pigment epithelium; sVLDLR, soluble ectodomain of VLDLR; VLDLR, very low-density lipoprotein receptor; VLDLRII, VLDLR variant II.

Article Snippet: Expression vectors for human ADAM10 (Addgene; plasmid #31717) and human ADAM17 (Addgene; plasmid #31713) were gifts from Dr Rik Derynck ( ).

Techniques: Knockdown, Plasmid Preparation, Over Expression, Transfection, Infection, Western Blot, Control, Modification, Variant Assay

Journal: The Journal of Biological Chemistry

Article Title: ADAM17 mediates ectodomain shedding of the soluble VLDL receptor fragment in the retinal epithelium

doi: 10.1016/j.jbc.2021.101185

Figure Lengend Snippet: CRISPR/Cas9-mediated ADAM17 KO blocked VLDLR shedding and its effect on Wnt signaling. A , representative images of Western blotting of sVLDLR in the media, and VLDLR, ADAM10, and ADAM17 in cell lysates of Ad-VLDLRII-infected WT and ADAM17 KO ARPE-19 cells. WT cells and ADAM17 KO cells were transduced with Ad-VLDLRII (MOI = 25) for 24 h. Then, culture media were replaced with serum-free DMEM for another 24 h. After that, the conditioned media and cell lysates were harvested for Western blot analysis. B , quantification of densitometry of sVLDLR in culture media normalized by VLDLR in cell lysates in ( A ) (n = 3). C , representative images of Western blotting of sVLDLR in culture media, and VLDLR, ADAM10, and ADAM17 in cell lysates in ADAM17 KO cells that were transfected with plasmid overexpressing ADAM17 (pADAM17) or RFP as control. D , quantification of densitometry of sVLDLR in culture media normalized by VLDLR in cell lysates in ( C ) (n = 3). In A and C , p and m indicated precursor and mature forms of ADAM10 and ADAM17, respectively. Data were presented as mean ± SD. ∗∗∗ p < 0.001. ADAM10, a disintegrin and metalloprotease 10; ADAM17, a disintegrin and metalloprotease 17; DMEM, Dulbecco's modified Eagle's medium; MOI, multiplicity of infection; RFP, red fluorescent protein; sVLDLR, soluble ectodomain of VLDLR; VLDLR, very low-density lipoprotein receptor.

Article Snippet: Expression vectors for human ADAM10 (Addgene; plasmid #31717) and human ADAM17 (Addgene; plasmid #31713) were gifts from Dr Rik Derynck ( ).

Techniques: CRISPR, Western Blot, Infection, Transduction, Transfection, Plasmid Preparation, Control, Modification

Journal: The Journal of Biological Chemistry

Article Title: ADAM17 mediates ectodomain shedding of the soluble VLDL receptor fragment in the retinal epithelium

doi: 10.1016/j.jbc.2021.101185

Figure Lengend Snippet: ADAM17 cleaved VLDLR in a cis manner, which was blocked by O-glycosylation. Primary human RPE cells were transfected with a plasmid expressing human ADAM17 (pADAM17) or RFP as control for 24 h. Cells were then infected with Ad-VLDLRII (MOI = 25) or RFP as control for another 24 h. After that, the cells were equally split into new 10-cm dishes for comparing cis and trans manner. A , representative images of Western blotting of sVLDLR in culture media, VLDLR, ADAM10, and ADAM17 in ARPE-19 cell lysates. Cis indicated that ADAM17 and VLDLR were overexpressed in same cells. Trans indicated that ADAM17 and VLDLR were overexpressed in different cells. B , densitometry analysis of sVLDLR in culture media normalized by the full-length VLDLR in cell lysates in ( A ) (n = 3). C , representative images of Western blotting of sVLDLR in culture media, and VLDLR and ADAM17 in O-glycosylation–deficient cells (ldlD). ldlD cells were transfected with the ADAM17 expression plasmid (pADAM17) or RFP as control for 24 h. Then, cells were transduced with adenovirus overexpressing VLDLRI, the full-length VLDLR with O-glycosylation domain (MOI = 25) or RFP as control for another 24 h. After that, culture media were replaced with a serum-free media with or without 10 μM d -(+)-galactose and 100 μM N -acetyl- d -galactosamine for 24 h. D , densitometry analysis of sVLDLR in culture media normalized by VLDLR in cell lysates in ( C ) (n = 3). Gal: d -(+)-galactose and N -acetyl- d -galactosamine. In A and C , p and m indicated precursor and mature forms of ADAM10 and ADAM17, respectively. Data were presented as mean ± SD. ∗ p < 0.05, ∗∗∗ p < 0.001. ADAM10, a disintegrin and metalloprotease 10; ADAM17, a disintegrin and metalloprotease 17; MOI, multiplicity of infection; RFP, red fluorescent protein; RPE, retinal pigment epithelium; VLDLR, very low-density lipoprotein receptor; VLDLRI, VLDLR variant I; VLDLRII, VLDLR variant II.

Article Snippet: Expression vectors for human ADAM10 (Addgene; plasmid #31717) and human ADAM17 (Addgene; plasmid #31713) were gifts from Dr Rik Derynck ( ).

Techniques: Glycoproteomics, Transfection, Plasmid Preparation, Expressing, Control, Infection, Western Blot, Transduction, Variant 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: Signal Transduction and Targeted Therapy

Article Title: Alpha hemolysin enhances the immune response by modulating dendritic cell differentiation via ADAM10-Notch signaling

doi: 10.1038/s41392-025-02432-3

Figure Lengend Snippet: Hla H35A improves the antibody titer and protection efficacy of antigens carried via ADAM10. a Experimental design for the mouse immunization model. b Serum levels of PA0833-specific IgG in mice immunized with PBS, PA0833, HPF or HPF with GI254023X after three immunizations ( n = 6–10 per group). The data were pooled from two independent experiments. c Serum levels of PA0833-specific IgG1 or IgG2a in mice immunized with PBS, PA0833, HPF or HPF with GI254023X treatment ( n = 6–10 per group). The data were pooled from two independent experiments. d Bacterial loads in the lungs were determined 48 h after challenge with a sublethal dose (3 × 10 6 CFU) of PAO1 in mice immunized with PBS, PA0833, HPF or HPF with GI254023X treatment ( n = 6–10 per group). The data were pooled from two independent experiments. e Body weight was measured 7 d after challenge with a sublethal dose (3 × 10 6 CFU) of PAO1 in mice immunized with PBS, PA0833, HPF or HPF with GI254023X treatment ( n = 7 per group). f Representative H&E staining of lung sections obtained 48 h after challenge with a sublethal dose (3 × 10 6 CFU) of PAO1 from mice immunized with PBS, PA0833, HPF or HPF with GI254023X treatment ( n = 3 per group). g TNF-α, IL-1β, IL-6, and IL-10 levels in the lungs were determined 48 h after challenge with a sublethal dose (3 × 10 6 CFU) of PAO1 in mice immunized with PBS, PA0833, HPF or HPF with GI254023X treatment ( n = 6–10 per group). The data were pooled from two independent experiments. h Kaplan–Meier survival curves of mice challenged with a lethal dose (1 × 10⁷ CFU) of PAO1, measured 14 days post-infection, following immunization with PBS, PA0833, HPF, or HPF with GI254023X treatment ( n = 15 per group). Each data point indicates a biological replicate in ( b – d ) and ( g ). The data are presented as the means ± s.e.m.s. Statistical significance was determined via one-way ANOVA followed by Tukey’s multiple comparisons test in ( b – d ) and ( g ). H&E staining hemoglobin and eosin staining, CFU colony forming unit

Article Snippet: Anti-ADAM10 antibody (1:250, Santa Cruz, Cat# sc-48400) was then added to the cells, which were then incubated overnight at 4 °C.

Techniques: Staining, Infection

Journal: Signal Transduction and Targeted Therapy

Article Title: Alpha hemolysin enhances the immune response by modulating dendritic cell differentiation via ADAM10-Notch signaling

doi: 10.1038/s41392-025-02432-3

Figure Lengend Snippet: Hla H35A promotes Fl-BMDC maturation via ADAM10. a Predicted accurate model of the interaction of HPFs with mouse ADAM10 (pLDDT = 72.7). The predicted local distance difference test score (pLDDT) was used to evaluate per-residue confidence, with values ≥ 70 indicating reliable backbone modeling. b Representative fluorescence images showing interactions of PA0833 or HPF with ADAM10 on the surface of Fl-BMDCs after 6 h of treatment with PA0833, HPF, or HPF with GI254023X ( n = 3 per group). The antigens used were PA0833 or HPF. The arrowheads indicate cells that captured the antigens. c Representative flow cytometry histogram (left) and quantification (right) of MHCII + cell frequencies gated on CD11c + cells from Fl-BMDCs after 7.5 h of treatment with PBS, PA0833, HPF, or HPF with GI254023X ( n = 6 per group). The data were pooled from two independent experiments. d Representative flow cytometry histogram (left) and quantification (right) of CD40 + cell frequencies gated on CD11c + cells from Fl-BMDCs after 7.5 h of treatment with PBS, PA0833, HPF, or HPF with GI254023X ( n = 6 per group). The data were pooled from two independent experiments. e Representative flow cytometry histogram (left) and quantification (right) of CD80 + cell frequencies gated on CD11c + cells from Fl-BMDCs after 7.5 h of treatment with PBS, PA0833, HPF, or HPF with GI254023X ( n = 6 per group). The data were pooled from two independent experiments. f Representative flow cytometry histogram (left) and quantification (right) of CD86 + cell frequencies gated on CD11c + cells from Fl-BMDCs after 7.5 h of treatment with PBS, PA0833, HPF, or HPF with GI254023X ( n = 6 per group). The data were pooled from two independent experiments. g Serum levels of TNF-α or IL-6 secreted from Fl-BMDCs after 7.5 h of treatment with PBS, PA0833, HPF, or HPF with GI254023X ( n = 6 per group). The data were pooled from two independent experiments. Each data point indicates a biological replicate in ( c – g ). The data are presented as the means ± s.e.m.s. Statistical significance was tested via one-way ANOVA followed by Tukey’s multiple comparisons test in ( c – g ). Fl-BMDCs Flt3L-induced bone marrow dendritic cells, pLDDT predicted local-distance difference test

Article Snippet: Anti-ADAM10 antibody (1:250, Santa Cruz, Cat# sc-48400) was then added to the cells, which were then incubated overnight at 4 °C.

Techniques: Residue, Fluorescence, Flow Cytometry

Journal: Signal Transduction and Targeted Therapy

Article Title: Alpha hemolysin enhances the immune response by modulating dendritic cell differentiation via ADAM10-Notch signaling

doi: 10.1038/s41392-025-02432-3

Figure Lengend Snippet: Hla H35A activates Notch signaling via ADAM10 in Fl-BMDCs. a Representative western blot (left) and quantification (right) of N1ICD, N2ICD, Notch3, or Notch4 in Fl-BMDCs after 4.5 h of treatment with PBS, PA0833, HPF, or HPF with GI254023X ( n = 3 per group). b Quantification of Hey1 , Hes1 , or Hes5 mRNA expression by qPCR in Fl-BMDCs after 6 h of treatment with PBS, PA0833, HPF, or HPF with GI254023X ( n = 3 per group). c Quantification of Adam10 , Notch1 , Notch2 , Notch3 , or Notch4 mRNA expression by qPCR in Fl-BMDCs after 4.5 h of treatment with PBS, PA0833, HPF, or HPF with GI254023X ( n = 3 per group). d TPMs of the Notch1 , Notch2 , Notch3 , or Notch4 genes in unstimulated Fl-BMDCs from bulk RNA-seq ( n = 3 per group) . e Quantification of Notch1 , Notch2 , Notch3 , or Notch4 mRNA expression by qPCR in Fl-BMDCs under homeostatic conditions ( n = 3 per group). Each data point indicates a biological replicate in ( a ) and ( d ). Each data point indicates a biological replicate derived from the average of three technical replicates in ( b , c and e ). The data are presented as the means ± s.e.m.s. Statistical significance was tested via one-way ANOVA followed by Tukey’s multiple comparisons test in ( a – e )

Article Snippet: Anti-ADAM10 antibody (1:250, Santa Cruz, Cat# sc-48400) was then added to the cells, which were then incubated overnight at 4 °C.

Techniques: Western Blot, Expressing, RNA Sequencing, Derivative Assay

Journal: Signal Transduction and Targeted Therapy

Article Title: Alpha hemolysin enhances the immune response by modulating dendritic cell differentiation via ADAM10-Notch signaling

doi: 10.1038/s41392-025-02432-3

Figure Lengend Snippet: Hla H35A induces Notch2-dependent cDC2s via ADAM10-Notch signaling. a Representative flow cytometry histogram (left) and quantification (right) of MHCII + cell frequencies gated on CD11c + cells from Fl-BMDCs after 7.5 h of treatment with PBS, PA0833, HPF, HPF with GI254023X or HPF with DAPT ( n = 6 per group). The data were pooled from two independent experiments. b Quantification of Ccr7 mRNA expression by qPCR in Fl-BMDCs after 7.5 h of treatment with PBS, PA0833, HPF, HPF with GI254023X or HPF with DAPT ( n = 3). c Representative flow cytometry plots (left) and quantification (right) of cDC subset frequencies in Fl-BMDCs after 12 h of treatment with PBS, PA0833, HPF, HPF with GI254023X or HPF with DAPT ( n = 6 per group). The data were pooled from two independent experiments. d Quantification of cDC1-associated Toll-like receptor mRNA expression by qPCR in Fl-BMDCs after 6 h of treatment with PBS, PA0833, HPF, HPF with GI254023X or HPF with DAPT ( n = 3). e Quantification of cDC2-associated Toll-like receptor mRNA expression by qPCR in Fl-BMDCs after 6 h of treatment with PBS, PA0833, HPF, HPF with GI254023X or HPF with DAPT ( n = 3). f Quantification of Irf4 or Klf4 mRNA expression by qPCR in Fl-BMDCs after 6 h of treatment with PBS, PA0833, HPF, HPF with GI254023X or HPF with DAPT ( n = 3). g Representative flow cytometry histogram (left) and quantification (right) of ESAM + cell frequencies gated on CD11c + MHCII + SIRPa + cells in Fl-BMDCs after 12 h of treatment with PBS, PA0833, HPF, HPF with GI254023X or HPF with DAPT ( n = 6 per group). The data were pooled from two independent experiments. h Serum levels of IL-23 secreted from Fl-BMDCs after 12 h of treatment with PBS, PA0833, HPF, HPF with GI254023X or HPF with DAPT ( n = 6 per group). The data were pooled from two independent experiments. Each data point indicates a biological replicate in ( a , c , g , and h ). Each data point indicates a biological replicate derived from the average of three technical replicates in ( b , d , e , and f ). The data are presented as the means ± s.e.m.s. Statistical significance was tested via one-way ANOVA followed by Tukey’s multiple comparisons test ( a , b , f , g and h ) and two-way ANOVA followed by Tukey’s multiple comparisons test ( c – e )

Article Snippet: Anti-ADAM10 antibody (1:250, Santa Cruz, Cat# sc-48400) was then added to the cells, which were then incubated overnight at 4 °C.

Techniques: Flow Cytometry, Expressing, Derivative Assay

Journal: Signal Transduction and Targeted Therapy

Article Title: Alpha hemolysin enhances the immune response by modulating dendritic cell differentiation via ADAM10-Notch signaling

doi: 10.1038/s41392-025-02432-3

Figure Lengend Snippet: Hla H35A promotes optimal Th17 and Tfh responses in dLNs by activating ADAM10-Notch signaling in cDCs. a Experimental design for the BMDC transfer model. Bone marrow (BM) cells from donor mice were cultured, stimulated and then transferred to recipient mice. The dLN and spleen of the recipient mice were analyzed on day 6 after transfer. Each mouse was intramuscularly injected with 5 × 10 5 CD11c + MHCII hi cells. b Numbers of cytokine-expressing CD4 + T cells in the dLNs on day 6 in mice that received Fl-BMDCs after 24 h of treatment with PBS, PA0833, HPF, HPF with GI254023X or HPF with DAPT ( n = 6 per group). The data were pooled from two independent experiments. c Frequencies of CD44 hi CD4 + T cells (left) and numbers of CD44 hi CD4 + T cells (right) in the dLNs on day 6 in mice that received Fl-BMDCs after 24 h of treatment with PBS, PA0833, HPF, GI254023X or HPF with DAPT ( n = 6 per group). The data were pooled from two independent experiments. d Frequencies of PD1 + CXCR5 + cells among CD44 hi CD4 + T cells (left) and numbers of PD1 + CXCR5 + CD44 hi CD4 + T cells (right) in the dLNs on day 6 in mice that received Fl-BMDCs after 24 h of treatment with PBS, PA0833, HPF, GI254023X or HPF with DAPT ( n = 6 per group). The data were pooled from two independent experiments. Each data point indicates a biological replicate in ( b – d ). The data are presented as the means ± s.e.m.s. Statistical significance was tested via one-way ANOVA followed by Tukey’s multiple comparisons test in ( b – d ). dLNs draining lymph nodes

Article Snippet: Anti-ADAM10 antibody (1:250, Santa Cruz, Cat# sc-48400) was then added to the cells, which were then incubated overnight at 4 °C.

Techniques: Cell Culture, Injection, Expressing

Journal: Signal Transduction and Targeted Therapy

Article Title: Alpha hemolysin enhances the immune response by modulating dendritic cell differentiation via ADAM10-Notch signaling

doi: 10.1038/s41392-025-02432-3

Figure Lengend Snippet: Hla H35A improves the immunogenicity and protective efficacy of carried antigens by activating ADAM10-Notch signaling in cDCs. a Experimental design for the Fl-BMDC transfer model. Each mouse was intramuscularly injected with 5 × 10 5 CD11c + MHCII hi cells. b Serum PA0833-specific IgG levels in mice following two adoptive transfers of Fl-BMDCs treated for 24 h with PBS, PA0833, HPF, HPF with GI254023X, or HPF with DAPT ( n = 8–9 per group). The data were pooled from two independent experiments. c Serum PA0833-specific IgG1 or IgG2a levels in mice following two adoptive transfers of Fl-BMDCs treated for 24 h with PBS, PA0833, HPF, HPF with GI254023X, or HPF with DAPT ( n = 8–9 per group). The data were pooled from two independent experiments. d Bacterial loads in the lungs determined 48 h after challenge with a sublethal dose (3 × 10 6 CFU) of PAO1 in mice following two adoptive transfers of Fl-BMDCs treated for 24 h with PBS, PA0833, HPF, HPF with GI254023X, or HPF with DAPT ( n = 8–9 per group). The data were pooled from two independent experiments. e Body weight was measured 7 d after challenge with a sublethal dose (3 × 10 6 CFU) of PAO1 in mice following two adoptive transfers of Fl-BMDCs treated for 24 h with PBS, PA0833, HPF, HPF with GI254023X, or HPF with DAPT ( n = 7 per group). f Representative H&E staining of lung sections obtained 48 h after challenge with a sublethal dose (3 × 10 6 CFU) of PAO1 from mice following two adoptive transfers of Fl-BMDCs treated for 24 h with PBS, PA0833, HPFs with GI254023X, or HPFs with DAPT ( n = 3 per group). g TNF-α, IL-1β, IL-6, and IL-10 levels in the lungs were determined 48 h after challenge with a sublethal dose (3 × 10 6 CFU) of PAO1 in mice following two adoptive transfers of Fl-BMDCs treated for 24 h with PBS, PA0833, HPF, GI254023X, or HPF with DAPT ( n = 8–9 per group). The data were pooled from two independent experiments. h Kaplan–Meier survival curves of mice challenged with a lethal dose (1 × 10 7 CFU) of PAO1, measured 14 days post-infection, following two adoptive transfers of Fl-BMDCs treated for 24 h with PBS, PA0833, HPF, HPF with GI254023X, or HPF with DAPT ( n = 10 per group). i Schematic diagram of the proposed mechanism by which Hla H35A enhances immunoprotection via ADAM10 on the surface of DCs. Each data point indicates a biological replicate in ( b – d and g ). The data are presented as the means ± s.e.m.s. Statistical significance was tested via one-way ANOVA followed by Tukey’s multiple comparisons test in ( b – d and g )

Article Snippet: Anti-ADAM10 antibody (1:250, Santa Cruz, Cat# sc-48400) was then added to the cells, which were then incubated overnight at 4 °C.

Techniques: Immunopeptidomics, Injection, Staining, Infection

Journal: Signal Transduction and Targeted Therapy

Article Title: Alpha hemolysin enhances the immune response by modulating dendritic cell differentiation via ADAM10-Notch signaling

doi: 10.1038/s41392-025-02432-3

Figure Lengend Snippet: Hla H35A induces human MoDC maturation via ADAM10. a Predicted model of the interaction of HPF with human ADAM10 (pLDDT = 73.6). The predicted local distance difference test score (pLDDT) was used to evaluate per-residue confidence, with values ≥ 70 indicating reliable backbone modeling. b Representative flow cytometry histogram (left) and quantification (right) of MHCII + cell frequencies gated on CD11c + cells from Fl-BMDCs after 6 h of treatment with PBS, PA0833, HPF, or HPF with GI254023X ( n = 3 per group). c Representative flow cytometry histogram (left) and quantification (right) of CD40 + cell frequencies gated on CD11c + cells from Fl-BMDCs after 6 h of treatment with PBS, PA0833, HPF, or HPF followed by GI254023X ( n = 3 per group). d Representative flow cytometry histogram (left) and quantification (right) of CD80 + cell frequencies gated on CD11c + cells from Fl-BMDCs after 6 h of treatment with PBS, PA0833, HPF, or HPF with GI254023X ( n = 3 per group). e Representative flow cytometry histogram (left) and quantification (right) of CD86 + cell frequencies gated on CD11c + cells from Fl-BMDCs after 6 h of treatment with PBS, PA0833, HPF, or HPF with GI254023X ( n = 3 per group). f Quantification of Tnfa or Il6 mRNA expression by qPCR in Fl-BMDCs after 6 h of treatment with PBS, PA0833, HPF, or HPF with GI254023X ( n = 3 per group). g Quantification of Hes1, Hes5 , or Hey1 mRNA expression by qPCR in Fl-BMDCs after 6 h of treatment with PBS, PA0833, HPF, or HPF with GI254023X ( n = 3 per group). Each data point indicates a biological replicate in ( b – e ). Each data point indicates a biological replicate derived from the average of three technical replicates in ( f and g ). The data are presented as the means ± s.e.m.s. Statistical significance was tested via one-way ANOVA followed by Tukey’s multiple comparisons test in ( b – g )

Article Snippet: Anti-ADAM10 antibody (1:250, Santa Cruz, Cat# sc-48400) was then added to the cells, which were then incubated overnight at 4 °C.

Techniques: Residue, Flow Cytometry, Expressing, Derivative Assay