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Exosome Diagnostics adam10 inhibitors
Adam10 Inhibitors, supplied by Exosome Diagnostics, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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adam10 inhibitors - by Bioz Stars, 2026-05

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ADAM10 and ADAM17 sheddases promote the release of SEVs. (a) NTA quantification of SEV concentrated by serial centrifugations from the cell culture medium of an equal number of 1C11 cells treated or not with the inhibitors of ADAM17 (TAPI‐2, 25 µM) or ADAM10 (GI254023X, 1 µM) for 96 h ( n = 6). (b) Protein level of SEVs isolated from the cell culture medium of 1C11 cells treated or not with ADAM inhibitors ( n = 4) as in (a) or silenced for ADAM17 or ADAM10 expression (siADAM10/17) for 48 h ( n = 3). (c). Confocal microscopy images of cell surface CD63 in 1C11 cells treated or not with TAPI‐2 (100 µM) or GI254023X (1 µM) for 24 h. Dotted lines show cell contour. Scale bar = 20 µm. (d) Biotin‐based assay, western blot analysis and quantification histogram of cell surface CD63 and CD81 in 1C11 cells treated or not with TAPI‐2 (100 µM) or GI254023X (1 µM) for 24 h ( n = 3). Tubulin in input lysates is shown as a loading control. (e) Protein level of SEVs isolated from the culture medium of an equal number of serotonergic 1C11 5‐HT neuronal cells ( n = 4), THP‐1 cells ( n = 3) or HeLa cells ( n = 3), and quantification of NanoLuc‐Hsp70 EVs luminescence in the cell culture medium of an equal number of genetically modified HeLa cells ( n = 5). 1C11 5‐HT , THP‐1 and HeLa cells were treated or not with TAPI‐2 (25 µM) or GI254023X (1 µM) for 96 h. NanoLuc‐Hsp70 HeLa cells were treated or not with TAPI‐2 (100 µM), GI254023X (1 µM), or silenced for ADAM17 or ADAM10 expression (siADAM10/17) for 24 h. Values are means ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 versus untreated cells (control, Ctrl) or siScramble‐treated cells.

Journal: Journal of Extracellular Vesicles

Article Title: ADAM Sheddase Activity Promotes the Detachment of Small Extracellular Vesicles From the Plasma Membrane

doi: 10.1002/jev2.70114

Figure Lengend Snippet: ADAM10 and ADAM17 sheddases promote the release of SEVs. (a) NTA quantification of SEV concentrated by serial centrifugations from the cell culture medium of an equal number of 1C11 cells treated or not with the inhibitors of ADAM17 (TAPI‐2, 25 µM) or ADAM10 (GI254023X, 1 µM) for 96 h ( n = 6). (b) Protein level of SEVs isolated from the cell culture medium of 1C11 cells treated or not with ADAM inhibitors ( n = 4) as in (a) or silenced for ADAM17 or ADAM10 expression (siADAM10/17) for 48 h ( n = 3). (c). Confocal microscopy images of cell surface CD63 in 1C11 cells treated or not with TAPI‐2 (100 µM) or GI254023X (1 µM) for 24 h. Dotted lines show cell contour. Scale bar = 20 µm. (d) Biotin‐based assay, western blot analysis and quantification histogram of cell surface CD63 and CD81 in 1C11 cells treated or not with TAPI‐2 (100 µM) or GI254023X (1 µM) for 24 h ( n = 3). Tubulin in input lysates is shown as a loading control. (e) Protein level of SEVs isolated from the culture medium of an equal number of serotonergic 1C11 5‐HT neuronal cells ( n = 4), THP‐1 cells ( n = 3) or HeLa cells ( n = 3), and quantification of NanoLuc‐Hsp70 EVs luminescence in the cell culture medium of an equal number of genetically modified HeLa cells ( n = 5). 1C11 5‐HT , THP‐1 and HeLa cells were treated or not with TAPI‐2 (25 µM) or GI254023X (1 µM) for 96 h. NanoLuc‐Hsp70 HeLa cells were treated or not with TAPI‐2 (100 µM), GI254023X (1 µM), or silenced for ADAM17 or ADAM10 expression (siADAM10/17) for 24 h. Values are means ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 versus untreated cells (control, Ctrl) or siScramble‐treated cells.

Article Snippet: The ADAM10 inhibitor GI254023X (TOCRIS Bioscience, 3995) and the PDK1 inhibitor BX912 (Axon Medchem BV, 1130) were prepared as 20 mM stock solution dissolved in 90% DMSO.

Techniques: Cell Culture, Isolation, Expressing, Confocal Microscopy, Western Blot, Control, Genetically Modified

PDK1‐RSK2 and ERK1/2‐RSK2 signalling pathways exert opposite effects on ADAM10/17‐dependent SEV release. (a) Schematic of signalling effectors involved in ADAM10/17‐mediated release of SEVs and selective inhibitors. NTKD: N‐terminal kinase domain; CTKD: C‐terminal kinase domain (left). Protein level of SEVs released by an equal number of 1C11 cells treated or not with inhibitors of PDK1 (BX912, 1 µM), ADAM17 (TAPI‐2, 25 µM), ADAM10 (GI254023X, 1 µM), ERK1/2 (PD98059, 2 µM), RSK2 N‐terminal (SL0101, 4 µM) or RSK2 C‐terminal (FMK, 3 µM) for 96 h (middle, n = 4). Quantification of NanoLuc‐Hsp70 HeLa EVs luminescence in the culture medium of an equal number of genetically modified HeLa cells treated or not with BX912 (5 µM), TAPI‐2 (100 µM), GI254023X (1 µM), PD98059 (5 µM), SL0101 (40 µM) or FMK (15 µM) for 24 h (right, n = 4). (b) Immunofluorescent labelling and quantification histogram of ADAM17 at the cell surface of 1C11 cells. Scale bar = 50 µm. (c) Biotin‐based assay, western blot analysis and quantification histogram of mature ADAM17 at the surface of 1C11 cells ( n = 3). Tubulin in input lysates is shown as a loading control. (d) Cell surface ADAM17 activity in 1C11 cells. (e) Immunofluorescent labelling and quantification histogram of ADAM10 at the cell surface of 1C11 cells. Scale bar = 50 µm. (f) Biotin‐based assay, western blot analysis and quantification histogram of mature ADAM10 at the surface of 1C11 cells ( n = 3). Tubulin in input lysates is shown as a loading control. For experiments in (b) to (f), cells were treated or not with BX912 (1 µM), PD98059 (5 µM), SL0101 (40 µM) or FMK (15 µM) for 24 h. Values are means ± SEM. ** p < 0.01, *** p < 0.001 and **** p < 0.0001 versus untreated cells (control, Ctrl) or BX912‐ or SL0101‐treated cells.

Journal: Journal of Extracellular Vesicles

Article Title: ADAM Sheddase Activity Promotes the Detachment of Small Extracellular Vesicles From the Plasma Membrane

doi: 10.1002/jev2.70114

Figure Lengend Snippet: PDK1‐RSK2 and ERK1/2‐RSK2 signalling pathways exert opposite effects on ADAM10/17‐dependent SEV release. (a) Schematic of signalling effectors involved in ADAM10/17‐mediated release of SEVs and selective inhibitors. NTKD: N‐terminal kinase domain; CTKD: C‐terminal kinase domain (left). Protein level of SEVs released by an equal number of 1C11 cells treated or not with inhibitors of PDK1 (BX912, 1 µM), ADAM17 (TAPI‐2, 25 µM), ADAM10 (GI254023X, 1 µM), ERK1/2 (PD98059, 2 µM), RSK2 N‐terminal (SL0101, 4 µM) or RSK2 C‐terminal (FMK, 3 µM) for 96 h (middle, n = 4). Quantification of NanoLuc‐Hsp70 HeLa EVs luminescence in the culture medium of an equal number of genetically modified HeLa cells treated or not with BX912 (5 µM), TAPI‐2 (100 µM), GI254023X (1 µM), PD98059 (5 µM), SL0101 (40 µM) or FMK (15 µM) for 24 h (right, n = 4). (b) Immunofluorescent labelling and quantification histogram of ADAM17 at the cell surface of 1C11 cells. Scale bar = 50 µm. (c) Biotin‐based assay, western blot analysis and quantification histogram of mature ADAM17 at the surface of 1C11 cells ( n = 3). Tubulin in input lysates is shown as a loading control. (d) Cell surface ADAM17 activity in 1C11 cells. (e) Immunofluorescent labelling and quantification histogram of ADAM10 at the cell surface of 1C11 cells. Scale bar = 50 µm. (f) Biotin‐based assay, western blot analysis and quantification histogram of mature ADAM10 at the surface of 1C11 cells ( n = 3). Tubulin in input lysates is shown as a loading control. For experiments in (b) to (f), cells were treated or not with BX912 (1 µM), PD98059 (5 µM), SL0101 (40 µM) or FMK (15 µM) for 24 h. Values are means ± SEM. ** p < 0.01, *** p < 0.001 and **** p < 0.0001 versus untreated cells (control, Ctrl) or BX912‐ or SL0101‐treated cells.

Article Snippet: The ADAM10 inhibitor GI254023X (TOCRIS Bioscience, 3995) and the PDK1 inhibitor BX912 (Axon Medchem BV, 1130) were prepared as 20 mM stock solution dissolved in 90% DMSO.

Techniques: Genetically Modified, Western Blot, Control, Activity Assay

ADAM10/17‐mediated cell release of SEVs is associated with the cleavage of several adhesion proteins at the SEV membrane. (a) Western blot of CD63 and tetherin performed with 1C11 cell lysates and concentrated SEVs. (b) Western blot of CADM1 performed with 1C11 cell lysates and concentrated SEVs. From left to right were loaded equal cell lysate protein amounts, SEVs concentrated from 10 6 cells (SEV per 10 6 cells), and equal SEV protein amounts (SEV/µg prot). CADM1 was detected using a C‐terminal or N‐terminal targeting antibody. CADM1 αCTF was quantified in SEVs ( n = 3). (c) Immunofluorescent labelling of CADM1 at the surface of 1C11 cells treated or not with TAPI‐2 (100 µM), GI254023X (1 µM) for 24 h, and related quantification histogram ( n = 3). Scale bar = 50 µm. (d) Western blot of E‐Cadherin performed with 1C11 cell lysates and concentrated SEVs. From left to right were loaded equal cell lysate protein amounts, SEVs purified from 10 6 cells (SEV per 10 6 cells), and equal SEV protein amounts (SEV/µg prot). E‐Cadherin αCTF was quantified in SEVs ( n = 3). (e) Immunofluorescent labelling of E‐Cadherin at the surface of 1C11 cells treated or not with TAPI‐2 (100 µM), GI254023X (1 µM) for 24 h and related quantification histogram ( n = 3). Scale bar = 50 µm. For experiments (a), (b) and (d), 1C11 cells were treated or not with TAPI‐2 (25 µM) or GI254023X (1 µM) for 96 h. Tubulin was used as a loading control. All western blot lanes derive from the same running blot but are shown separately. Values are means ± SEM. * p < 0.05, ** p < 0.01 and **** p < 0.0001 versus untreated cells (control, Ctrl).

Journal: Journal of Extracellular Vesicles

Article Title: ADAM Sheddase Activity Promotes the Detachment of Small Extracellular Vesicles From the Plasma Membrane

doi: 10.1002/jev2.70114

Figure Lengend Snippet: ADAM10/17‐mediated cell release of SEVs is associated with the cleavage of several adhesion proteins at the SEV membrane. (a) Western blot of CD63 and tetherin performed with 1C11 cell lysates and concentrated SEVs. (b) Western blot of CADM1 performed with 1C11 cell lysates and concentrated SEVs. From left to right were loaded equal cell lysate protein amounts, SEVs concentrated from 10 6 cells (SEV per 10 6 cells), and equal SEV protein amounts (SEV/µg prot). CADM1 was detected using a C‐terminal or N‐terminal targeting antibody. CADM1 αCTF was quantified in SEVs ( n = 3). (c) Immunofluorescent labelling of CADM1 at the surface of 1C11 cells treated or not with TAPI‐2 (100 µM), GI254023X (1 µM) for 24 h, and related quantification histogram ( n = 3). Scale bar = 50 µm. (d) Western blot of E‐Cadherin performed with 1C11 cell lysates and concentrated SEVs. From left to right were loaded equal cell lysate protein amounts, SEVs purified from 10 6 cells (SEV per 10 6 cells), and equal SEV protein amounts (SEV/µg prot). E‐Cadherin αCTF was quantified in SEVs ( n = 3). (e) Immunofluorescent labelling of E‐Cadherin at the surface of 1C11 cells treated or not with TAPI‐2 (100 µM), GI254023X (1 µM) for 24 h and related quantification histogram ( n = 3). Scale bar = 50 µm. For experiments (a), (b) and (d), 1C11 cells were treated or not with TAPI‐2 (25 µM) or GI254023X (1 µM) for 96 h. Tubulin was used as a loading control. All western blot lanes derive from the same running blot but are shown separately. Values are means ± SEM. * p < 0.05, ** p < 0.01 and **** p < 0.0001 versus untreated cells (control, Ctrl).

Article Snippet: The ADAM10 inhibitor GI254023X (TOCRIS Bioscience, 3995) and the PDK1 inhibitor BX912 (Axon Medchem BV, 1130) were prepared as 20 mM stock solution dissolved in 90% DMSO.

Techniques: Membrane, Western Blot, Purification, Control

Scheme representation of ADAM10/17 role in cell release of SEVs. ADAM10/17 promotes the detachment of SEVs from the cell surface by catalysing the cleavage of adhesion proteins (CADM1, E‐Cadherin…) present at the membrane of SEVs. ADAM10/17‐mediated basal release of SEVs depends on a balanced control of 3‐phosphoinositide‐dependent kinase 1 (PDK1) and ERK1/2 signalling pathways converging on 90‐kDa ribosomal S6 kinase‐2 (RSK2), which, in turn, fine‐tunes ADAM17 bioavailability and ADAM10/17 enzymatic activities at the plasma membrane (left). When the PDK1‐RSK2 pathway is inhibited with BX912, the release of SEVs is amplified due to the positive action of ERK1/2‐RSK2 signalling on plasma membrane ADAM shedding activity (middle). When the ERK1/2‐RSK2 pathway is inhibited with PD98059, the release of SEVs is attenuated due to the negative action of PDK1‐RSK2 signalling on plasma membrane ADAM shedding activity (right). CTKD, C‐terminal kinase domain of RSK2; MVB, multivesicular body; NTKD, N‐terminal kinase domain of RSK2.

Journal: Journal of Extracellular Vesicles

Article Title: ADAM Sheddase Activity Promotes the Detachment of Small Extracellular Vesicles From the Plasma Membrane

doi: 10.1002/jev2.70114

Figure Lengend Snippet: Scheme representation of ADAM10/17 role in cell release of SEVs. ADAM10/17 promotes the detachment of SEVs from the cell surface by catalysing the cleavage of adhesion proteins (CADM1, E‐Cadherin…) present at the membrane of SEVs. ADAM10/17‐mediated basal release of SEVs depends on a balanced control of 3‐phosphoinositide‐dependent kinase 1 (PDK1) and ERK1/2 signalling pathways converging on 90‐kDa ribosomal S6 kinase‐2 (RSK2), which, in turn, fine‐tunes ADAM17 bioavailability and ADAM10/17 enzymatic activities at the plasma membrane (left). When the PDK1‐RSK2 pathway is inhibited with BX912, the release of SEVs is amplified due to the positive action of ERK1/2‐RSK2 signalling on plasma membrane ADAM shedding activity (middle). When the ERK1/2‐RSK2 pathway is inhibited with PD98059, the release of SEVs is attenuated due to the negative action of PDK1‐RSK2 signalling on plasma membrane ADAM shedding activity (right). CTKD, C‐terminal kinase domain of RSK2; MVB, multivesicular body; NTKD, N‐terminal kinase domain of RSK2.

Article Snippet: The ADAM10 inhibitor GI254023X (TOCRIS Bioscience, 3995) and the PDK1 inhibitor BX912 (Axon Medchem BV, 1130) were prepared as 20 mM stock solution dissolved in 90% DMSO.

Techniques: Membrane, Control, Clinical Proteomics, Amplification, Activity Assay

$Inclusion of the Ly6G/C-specific VHH16 enhances the activation of the conditional antimicrobial therapeutic peptide. (A) Design of a conditional antimicrobial therapeutic for delivery of POL. (B) Characterization of the POL conjugate VHH16-(ABD) 2 -(EEG) 6 -S17-POL (left: SDS-PAGE; Coomassie blue staining, right: analysis by MALDI-ToF MS reported as mass-to-charge ratio m / z ). (C) In vitro cleavage assay of VHH16-(ABD) 2 -(EEG) 6 -S17-POL-Cy7 by ADAM10 detected via Cy7 fluorescence using an Odyssey CLx imager. (D) In vitro evaluation of masking of antimicrobial activity by VHH16-(ABD) 2 -(EEG) 6 -S17-POL in a microdilution assay on PAO1. Bacterial viabilities were measured based on OD600 absorbance measurements normalized to the untreated control. (E) Experimental timeline and workflow for in vivo evaluation of biodistribution and activation of POL-Cy7 conjugates. (F) Quantification of total and activated fractions of the POL-Cy7 conjugates in PAO1-infected lungs presented as % injected dose (ID)/ gram (g). Panels D and F were plotted as mean ± standard deviation (SD) ( n = 3). Panel F was analyzed with one-way ANOVA with Tukey post hoc tests. Selected comparisons between POL-Cy7 and released POL-Cy7 from the S17 conjugates were shown in pink. The asterisk (*) denotes statistical significance ( P < 0.05). Panel E was partly created with BioRender.com .$

Journal: ACS Nano

Article Title: Nanobody-Targeted Conditional Antimicrobial Therapeutics

doi: 10.1021/acsnano.4c16007

Figure Lengend Snippet: Inclusion of the Ly6G/C-specific VHH16 enhances the activation of the conditional antimicrobial therapeutic peptide. (A) Design of a conditional antimicrobial therapeutic for delivery of POL. (B) Characterization of the POL conjugate VHH16-(ABD) 2 -(EEG) 6 -S17-POL (left: SDS-PAGE; Coomassie blue staining, right: analysis by MALDI-ToF MS reported as mass-to-charge ratio m / z ). (C) In vitro cleavage assay of VHH16-(ABD) 2 -(EEG) 6 -S17-POL-Cy7 by ADAM10 detected via Cy7 fluorescence using an Odyssey CLx imager. (D) In vitro evaluation of masking of antimicrobial activity by VHH16-(ABD) 2 -(EEG) 6 -S17-POL in a microdilution assay on PAO1. Bacterial viabilities were measured based on OD600 absorbance measurements normalized to the untreated control. (E) Experimental timeline and workflow for in vivo evaluation of biodistribution and activation of POL-Cy7 conjugates. (F) Quantification of total and activated fractions of the POL-Cy7 conjugates in PAO1-infected lungs presented as % injected dose (ID)/ gram (g). Panels D and F were plotted as mean ± standard deviation (SD) ( n = 3). Panel F was analyzed with one-way ANOVA with Tukey post hoc tests. Selected comparisons between POL-Cy7 and released POL-Cy7 from the S17 conjugates were shown in pink. The asterisk (*) denotes statistical significance ( P < 0.05). Panel E was partly created with BioRender.com .

Article Snippet: For the biodistribution study with VHH16 competition or ADAM10 protease inhibitor, either VHH16-(ABD) 2 -(EEG) 6 (10 equiv in 50 μL PBS) or the ADAM10-selective inhibitor GI254023X (MedChemExpress, NJ, U.S.A.) (5 mg/kg dose in 50 μL 0.9% NaCl (10% DMSO, 20% sulfobutylether-β-cyclodextrin)) was administered intratracheally at 5.5 h postinfection followed by intravenous treatment with VHH16-(ABD) 2 -(EEG) 6 -S17-POL-Cy7 (10 nmol) at 6 h postinfection.

Techniques: Activation Assay, SDS Page, Staining, In Vitro, Cleavage Assay, Fluorescence, Activity Assay, Microdilution Assay, Control, In Vivo, Infection, Injection, Standard Deviation

$Enhanced activation of a VHH16-targeted conditional antimicrobial therapeutic requires interaction with the Ly6G/C target as well as proteolytic activity of ADAM10. (A) Experimental timeline for in vivo evaluation of the biodistribution and activation of VHH16-(ABD) 2 -(EEG) 6 -Sx-POL-Cy7 with different cleavable linkers (Sx). (B) Quantification of total and activated fractions of the POL-Cy7 conjugates in PAO1-infected lungs presented as % ID/g. (C) Experimental timeline for in vivo evaluation of biodistribution and activation of VHH16-(ABD) 2 -(EEG) 6 -S17-POL-Cy7 using intratracheal pretreatment with an excess either of VHH16-(ABD) 2 -(EEG) 6 or of the ADAM10-selective inhibitor GI254023X. (D) Quantification of total and activated fractions of VHH16-(ABD) 2 -(EEG) 6 -S17-POL-Cy7 in PAO1-infected lungs presented as % ID/g. (E) Experimental timeline for analysis by flow cytometry of VHH16-(ABD) 2 -(EEG) 6 -NC-SulfoCy5 accumulation in different cell populations of PAO1-infected lungs. (F) A representative density plot of the lung cell populations based on in vivo accumulated VHH16 and ex vivo stained Ly6G using an anti-Ly6G monoclonal antibody. Gates were set based on the intensity of VHH16 (±) and Ly6G (hi/int/neg). (G) Quantification of each cell population presented as a percentage of the total live cell population. (H) Quantification of ADAM10 in each cell population based on ex vivo staining with an anti-ADAM10 monoclonal antibody, presented as median fluorescence intensity (MFI). Panels B, D, G, and H were plotted as mean ± SD ( n = 3). Panels B, D, and H were analyzed with one-way ANOVA with Tukey post hoc tests. Selected comparisons between released POL-Cy7 from the S17 conjugate and the other conjugates were shown in pink. The asterisk (*) denotes statistical significance ( P < 0.05).$

Journal: ACS Nano

Article Title: Nanobody-Targeted Conditional Antimicrobial Therapeutics

doi: 10.1021/acsnano.4c16007

Figure Lengend Snippet: Enhanced activation of a VHH16-targeted conditional antimicrobial therapeutic requires interaction with the Ly6G/C target as well as proteolytic activity of ADAM10. (A) Experimental timeline for in vivo evaluation of the biodistribution and activation of VHH16-(ABD) 2 -(EEG) 6 -Sx-POL-Cy7 with different cleavable linkers (Sx). (B) Quantification of total and activated fractions of the POL-Cy7 conjugates in PAO1-infected lungs presented as % ID/g. (C) Experimental timeline for in vivo evaluation of biodistribution and activation of VHH16-(ABD) 2 -(EEG) 6 -S17-POL-Cy7 using intratracheal pretreatment with an excess either of VHH16-(ABD) 2 -(EEG) 6 or of the ADAM10-selective inhibitor GI254023X. (D) Quantification of total and activated fractions of VHH16-(ABD) 2 -(EEG) 6 -S17-POL-Cy7 in PAO1-infected lungs presented as % ID/g. (E) Experimental timeline for analysis by flow cytometry of VHH16-(ABD) 2 -(EEG) 6 -NC-SulfoCy5 accumulation in different cell populations of PAO1-infected lungs. (F) A representative density plot of the lung cell populations based on in vivo accumulated VHH16 and ex vivo stained Ly6G using an anti-Ly6G monoclonal antibody. Gates were set based on the intensity of VHH16 (±) and Ly6G (hi/int/neg). (G) Quantification of each cell population presented as a percentage of the total live cell population. (H) Quantification of ADAM10 in each cell population based on ex vivo staining with an anti-ADAM10 monoclonal antibody, presented as median fluorescence intensity (MFI). Panels B, D, G, and H were plotted as mean ± SD ( n = 3). Panels B, D, and H were analyzed with one-way ANOVA with Tukey post hoc tests. Selected comparisons between released POL-Cy7 from the S17 conjugate and the other conjugates were shown in pink. The asterisk (*) denotes statistical significance ( P < 0.05).

Techniques: Activation Assay, Activity Assay, In Vivo, Infection, Flow Cytometry, Ex Vivo, Staining, Fluorescence

$Demonstration of VHH16-enhanced activation of a conditional antimicrobial therapeutic protein. (A) Design of a conditional antimicrobial therapeutic for delivery of PNT4. (B) In vitro cleavage assay of VHH16-ABD-(EEG) 6 -S17-PNT4-sulfo-Cy7 by ADAM10 detected via sulfo-Cy7 fluorescence using an Odyssey CLx imager. (C) In vitro evaluation of antimicrobial activity masking of VHH16-ABD-(EEG) 6 -S17-PNT4 via a microdilution assay on PAO1. Bacteria viabilities were measured based on OD600 absorbance normalized to the untreated control. (D) Experimental timeline for in vivo evaluation of the biodistribution and activation of VHH16-ABD-(EEG) 6 -S17-PNT4-sulfo-Cy7. Quantification of the total and activated fractions of PNT4-sulfo-Cy7 in (E) PAO1-infected lungs, (F) liver, and (G) kidneys presented as % ID/g. (H) Experimental timeline for in vivo evaluation of the therapeutic efficacy of VHH16-ABD-(EEG) 6 -S17-PNT4. (I) Quantification of bacterial burden from the treated lungs presented as log(cfu/g). The dotted line denotes the limit of detection. Panels C, E–G, and I were plotted as mean ± SD ( n = 3 for panels C and E–G; n = 5 for panel I). Panels E–G and I were analyzed with one-way ANOVA with Tukey post hoc tests. Selected comparisons between PNT4-sulfo-Cy7 and released PNT4-sulfo-Cy7 from the conditional therapeutics were shown in pink. The asterisk (*) denotes statistical significance ( P < 0.05). The evaluation of efficacy was confirmed in two independent studies with similar results.$

Journal: ACS Nano

Article Title: Nanobody-Targeted Conditional Antimicrobial Therapeutics

doi: 10.1021/acsnano.4c16007

Figure Lengend Snippet: Demonstration of VHH16-enhanced activation of a conditional antimicrobial therapeutic protein. (A) Design of a conditional antimicrobial therapeutic for delivery of PNT4. (B) In vitro cleavage assay of VHH16-ABD-(EEG) 6 -S17-PNT4-sulfo-Cy7 by ADAM10 detected via sulfo-Cy7 fluorescence using an Odyssey CLx imager. (C) In vitro evaluation of antimicrobial activity masking of VHH16-ABD-(EEG) 6 -S17-PNT4 via a microdilution assay on PAO1. Bacteria viabilities were measured based on OD600 absorbance normalized to the untreated control. (D) Experimental timeline for in vivo evaluation of the biodistribution and activation of VHH16-ABD-(EEG) 6 -S17-PNT4-sulfo-Cy7. Quantification of the total and activated fractions of PNT4-sulfo-Cy7 in (E) PAO1-infected lungs, (F) liver, and (G) kidneys presented as % ID/g. (H) Experimental timeline for in vivo evaluation of the therapeutic efficacy of VHH16-ABD-(EEG) 6 -S17-PNT4. (I) Quantification of bacterial burden from the treated lungs presented as log(cfu/g). The dotted line denotes the limit of detection. Panels C, E–G, and I were plotted as mean ± SD ( n = 3 for panels C and E–G; n = 5 for panel I). Panels E–G and I were analyzed with one-way ANOVA with Tukey post hoc tests. Selected comparisons between PNT4-sulfo-Cy7 and released PNT4-sulfo-Cy7 from the conditional therapeutics were shown in pink. The asterisk (*) denotes statistical significance ( P < 0.05). The evaluation of efficacy was confirmed in two independent studies with similar results.

Techniques: Activation Assay, In Vitro, Cleavage Assay, Fluorescence, Activity Assay, Microdilution Assay, Bacteria, Control, In Vivo, Infection

Effect of L-arginine on ADAM17 translocation to the cell surface in C2C12 cells. Results obtained by flow cytometry for control cells and those exposed for 2 h to L-arginine. (a) Delimitation of cellular P1, excluding remaining cellular debris by flow cytometry. (b) The cell quantity is observed on the vertical axis and the presence of FITC-A marker on the horizontal axis. (c) Fluorescence mean analysis performed by flow cytometry. The graphs shown correspond to a picture of a representative result ( n = 4).

Journal: Biochemistry Research International

Article Title: L-Arginine Activates the Neuregulin-1/ErbB Receptor Signaling Pathway and Increases Utrophin mRNA Levels in C2C12 Cells

doi: 10.1155/bri/2171745

Figure Lengend Snippet: Effect of L-arginine on ADAM17 translocation to the cell surface in C2C12 cells. Results obtained by flow cytometry for control cells and those exposed for 2 h to L-arginine. (a) Delimitation of cellular P1, excluding remaining cellular debris by flow cytometry. (b) The cell quantity is observed on the vertical axis and the presence of FITC-A marker on the horizontal axis. (c) Fluorescence mean analysis performed by flow cytometry. The graphs shown correspond to a picture of a representative result ( n = 4).

Article Snippet: ADAM17 and ADAM10 inhibitor (GW280264X) and ADAM10 inhibitor (GI254023X), from AOBIOUS INC, were kindly donated by Dr. Ricardo Moreno (Pontificia Universidad Católica de Chile).

Techniques: Translocation Assay, Flow Cytometry, Control, Marker, Fluorescence

Immunostaining of ADAM17 in C2C12 myotubes. Analysis by immunofluorescence (a) control and (b) myotubes stimulated by 2 h with L-arginine 5 mM. Cells were fixed and incubated with anti-ADAM17 antibody (1:100) and then with secondary antibody Alexa Fluor 546 donkey anti-rabbit IgG (H + L) (1:500). Images were obtained by a confocal microscope ( n = 3). Scale bar = 10 μm.

Journal: Biochemistry Research International

Article Title: L-Arginine Activates the Neuregulin-1/ErbB Receptor Signaling Pathway and Increases Utrophin mRNA Levels in C2C12 Cells

doi: 10.1155/bri/2171745

Figure Lengend Snippet: Immunostaining of ADAM17 in C2C12 myotubes. Analysis by immunofluorescence (a) control and (b) myotubes stimulated by 2 h with L-arginine 5 mM. Cells were fixed and incubated with anti-ADAM17 antibody (1:100) and then with secondary antibody Alexa Fluor 546 donkey anti-rabbit IgG (H + L) (1:500). Images were obtained by a confocal microscope ( n = 3). Scale bar = 10 μm.

Article Snippet: ADAM17 and ADAM10 inhibitor (GW280264X) and ADAM10 inhibitor (GI254023X), from AOBIOUS INC, were kindly donated by Dr. Ricardo Moreno (Pontificia Universidad Católica de Chile).

Techniques: Immunostaining, Immunofluorescence, Control, Incubation, Microscopy

Effect of L-arginine on utrophin gene expression in the presence of ADAMs inhibitors in C2C12 cells. The C2C12 myotubes were stimulated for 4 h with 5 mM L-arginine in the presence or absence of ADAM17 and ADAM10 inhibitors (GW280264X; Gw) and only ADAM10 (GI254023X; Gi). Then, using qPCR, the results were evaluated by the delta method Ct (2 −ΔΔCt ) between the average Ct of the gene of interest (utrophin) and the housekeeping gene (GAPDH). The results were expressed as a percentage of the corresponding nonstimulated control (4 h: 100%). Bars represent mean ± SEM ( n = 3). ⁣ ∗ p ≤ 0.05 versus correspondent control, ANOVA followed by Dunnett's multiple comparison test.

Journal: Biochemistry Research International

Article Title: L-Arginine Activates the Neuregulin-1/ErbB Receptor Signaling Pathway and Increases Utrophin mRNA Levels in C2C12 Cells

doi: 10.1155/bri/2171745

Figure Lengend Snippet: Effect of L-arginine on utrophin gene expression in the presence of ADAMs inhibitors in C2C12 cells. The C2C12 myotubes were stimulated for 4 h with 5 mM L-arginine in the presence or absence of ADAM17 and ADAM10 inhibitors (GW280264X; Gw) and only ADAM10 (GI254023X; Gi). Then, using qPCR, the results were evaluated by the delta method Ct (2 −ΔΔCt ) between the average Ct of the gene of interest (utrophin) and the housekeeping gene (GAPDH). The results were expressed as a percentage of the corresponding nonstimulated control (4 h: 100%). Bars represent mean ± SEM ( n = 3). ⁣ ∗ p ≤ 0.05 versus correspondent control, ANOVA followed by Dunnett's multiple comparison test.

Article Snippet: ADAM17 and ADAM10 inhibitor (GW280264X) and ADAM10 inhibitor (GI254023X), from AOBIOUS INC, were kindly donated by Dr. Ricardo Moreno (Pontificia Universidad Católica de Chile).

Techniques: Gene Expression, Control, Comparison

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