heparan sulfate  (AMS Biotechnology)

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. Volcano plot illustrating the proteins associated with GFP-SDC4 according to differential proteomics, with log2 fold change (x-axis) and -log10 (p-value) (y-axis). Data were obtained by GFP-pulldown from total extracts of MCF7 cells overexpressing GFP-SDC4 compared to MCF7 cells overexpressing GFP alone. Data result from three different experiments processed three times. SDC4 (the bait) is represented in green, ADAM10 and ADAM17, the two metalloproteases associated with SDC4 are represented in red. The other proteins are represented in grey. The proteins showing non-significant differences are represented below the dotted lines (threshold at 1 for the x-axis, difference =2 and 1.5 for y-axis; p-value = 0.3). B. Co-immunoprecipitation experiments from MCF7 cell lysates, confirming the association of endogenous ADAM10 and ADAM17 with GFP-SDC4, but not GFP. The GFP precipitates (IP) were subjected to Western blot with antibodies recognizing proteins as indicated on the right. C . Left: Scheme illustrating the complexity of SDC4 structure and processing. (Upper part) SDC4 full-length (FL) core protein (black) is substituted with glycosaminoglycan chains (GAG) of the heparan sulfate type (green). 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 easily detectable by immunoblotting. (Lower part) SDC4 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 SDC4 and SDC4 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 SDC4. Note that the FL form of SDC4 (SDC4-FL) abounds in cell lysates, while the CTF is less abundant. On the contrary, the SDC4-CTF is abundant and the SDC4-FL is barely detectable in sEVs. D. MCF7 cells downregulated for ADAM10 (siADAM10) or ADAM17 (siADAM17) and control cells (siCTRL) were evaluated for SDC4 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 < 0.001, n.s. non-significant). E . 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 SDC4 FL and CTF. Histograms represent the mean signal intensity for indicated proteins relative to the signal in control cells, ± SEM. ** P < 0.01 (unpaired non-parametric Mann–Whitney test).

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: Immunoprecipitation, Western Blot, SDS Page, Membrane, Control, Activity Assay, Isolation, MANN-WHITNEY

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

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-B. Western blot of the cell lysates and sEVs from HEK293 cells treated with the ADAM10 inhibitor GI254023X or DMSO as control, in the presence or absence of serum (FCS), show that ADAM10 supports the cleavage of various receptors, as indicated by the observed increase of non-cleaved receptors in the sEV of those cells, irrespectively of serum (although the absence of serum significantly reduces sEV accumulations). These control experiments were performed to ensure the pertinence of the endosomal escape assay as developed by Hyka et al., 2025 in the context of the present study. For SDCs (A), cells were treated with heparatinase and chondroitinase (GAG digestion +) or not (GAG digestion -). Heparan sulfate proteoglycans / SDCs are detected with the mAb 3G10 recognizing GAG chain stubs left after digestion ( David et al , 1992 ). C. Left. Histogram illustrating that the uptake of HiBiT-syntenin sEVs by HEK293 LgBiT cells after 4 hours of incubation is comparable regardless of treatment with the ADAM10 inhibitor GI254023X. Right. Histogram illustrating that endosomal escape of HiBit-syntenin sEVs is abolished when sEVs originate from HEK293 treated with ADAM10 inhibitor (GI254023X). Histograms represent mean signal intensities in relative light units (RLU) after addition of the nanoluciferase substrate Furimazine. D. Illustrative western blot of size exclusion chromatography (SEC) experiments for the preparation of sEVs for functional assays. Left. Syntenin, CD9, CD81, CD63, ADAM10, SDC1 and SDC4 were used as markers to select ad-hoc fractions from the conditioned media of MCF7 cells KO for ADAM10 (sEV KO) or control cells (sEV WT) as indicated. Fractions 6-9 were pooled before incubation with HUVEC cells. Right. Syntenin, CD9, CD81, CD63, SDC1 and EGFR were used as markers to select ad-hoc fractions from the conditioned media of MDA-MB-468 WT cells as indicated. Fractions 6-8 were pooled before incubation with HUVEC cells. E. The phosphorylation of selected proteins (top nine proteins showing increased phosphorylation after ADAM10-KO EV treatment) were determined using the phospho-array (R&D systems). Signals for each phosphorylated protein are presented as a pair of duplicate spots for the same exposition of 15 minutes. Average densitometric values for the phosphorylated proteins are shown in the heatmap . F. Heatmap showing log 2-fold changes of protein phosphorylation after treatment with sEV from MDA-MB-468 ADAM10 KO cells compared to control WT cells. The log 2-fold changes of protein phosphorylation after sEV KO treatment compared to control (sEV WT) were shown in the heatmap. The level of average densitometric values (Log2) are presented as a spectrum of color where white and red colors represent the lowest and the highest values in the row according to the indicated scale. The difference (diff, shown in ochre) corresponds to the difference between KO and WT conditions of the same protein, based on their respective signal intensity difference between KO and Ctrl conditions. The experiment was performed two or three times depending on the analyzed protein.

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: Western Blot, Control, Incubation, Size-exclusion Chromatography, Functional Assay, Phospho-proteomics