Journal: bioRxiv
Article Title: ADAM10 tailors extracellular vesicles for content transfer rather than signaling by contact
doi: 10.64898/2026.02.12.705562
Figure Lengend Snippet: A . Left: Scheme illustrating the complexity of SDC1 structure and processing. (Upper part) SDC1 full-length (FL) core protein (black) is substituted with glycosaminoglycan chains (GAG) of the heparan (green) and chondroitin (orange) sulfate type. The trimming of GAG chains by specific enzymes is necessary for the FL protein to migrate at a discrete band in SDS-PAGE and to be detectable by immunoblotting. (Lower part) SDC1 core protein can be cleaved by proteases generating two main fragments: an N-terminal fragment comprising most of the GAG-substituted extracellular domain (ECD) and a C-terminal fragment (CTF) comprising the remainder of the ECD, the membrane-spanning and the cytoplasmic domain. Right: Western blot illustrating the signals obtained, after GAG-digestion, for FL SDC1 and SDC1 CTF in the cells and the sEV enriched fraction obtained after differential ultracentrifugation of the conditioned extracellular media. Signals were obtained with an antibody recognizing the intracellular domain of SDC1. Note that the FL form of SDC1 (SDC1-FL) abounds in cell lysates, while the CTF is less abundant. On the contrary, the SDC1-CTF is abundant and the SDC1-FL is barely detectable in sEVs. B. MCF7 cells downregulated for ADAM10 (siADAM10) or ADAM17 (siADAM17) and control cells (siCTRL) were evaluated for SDC1 FL and CTF abundance in cells and sEVs by Western blot, after GAG-digestion. Histograms represent the mean signal intensity for indicated proteins relative to the signal in control cells, ± SEM. Statistical analysis was performed using the Kruskal-Wallis one-way non-parametric ANOVA test (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, n.s. non-significant). C. MCF7 cells downregulated for ADAM10 (siADAM10) and control cells (siCTRL) were treated with heparitinase and chondroitinase (GAG digestion +) or not (GAG digestion -) to evaluate SDC substitution with GAG chains. SDCs FL and CTF in cells and sEVs were analyzed by Western blot as indicated. Single blots examining the relative abundance of FL versus CTF forms of SDC1 ( D ) or SDC4 ( E ) forms are provided. F . sEVs secreted by MCF7 cells inhibited for ADAM10 activity, (GI254023X) versus controls (DMSO) were isolated from conditioned media by differential ultracentrifugation. sEVs were analyzed by Western blot after GAG-digestion to evaluate the levels of SDC1 FL and CTF. Histograms represent the mean signal intensity for indicated proteins relative to the signal in control cells, ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, n.s. non-significant (unpaired non-parametric Mann–Whitney test). n indicates the number of independent experiments; each point represents one independent experiment.
Article Snippet: To enzymatically digest the glycosaminoglycans (GAGs) on SDC, including heparan sulfate (HS) and chondroitin sulfate (CS), nine volumes of cell extract or EVs were incubated with one volume of 10x heparitinase reaction buffer (1M NaCl, 500mM hepes pH 7,0, 10mM CaCl2, 1% TX100, Pepstatin and Leupeptin, both at a concentration of 10 μg/mL), heparitinase (0.4 milliIU, amsbio), and chondroitinase (20milliunits, amsbio).
Techniques: SDS Page, Western Blot, Membrane, Control, Activity Assay, Isolation, MANN-WHITNEY
