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

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

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

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