Journal: Cell reports

Article Title: Transfer of Functional Cargo in Exomeres

doi: 10.1016/j.celrep.2019.01.009

Figure Lengend Snippet: (A) Exosomes and exomeres contain sialyltransferase activity. Varying amounts of DiFi cell-derived exosomes or exomeres were added to a sialyltransferase activity kit (R&D Systems), which measures the transfer of sialic acid from CMP-sialic acid to an acceptor substrate. Three independent biological experiments were performed, and data are presented as mean ± SEM. (B) Immunoblot analysis of ST6Gal-I levels in colon cancer cell lines. SW48 cells were stably transduced with control vector or with ST6Gal-I expression construct lentiviral particles. m, membrane; s, soluble. (C) ST6Gal-I in exomeres and exosomes is delivered to recipient cells. Exosomes and exomeres derived from DiFi cells were applied to SW948 and SW48 cells and cells were harvested at different time points. Lysates were either directly used for immunoblotting to detect ST6Gal-I (top), or incubated with agarose-conjugated Sambucus nigra agglutinin (SNA) lectin (bottom). α2,6-Sialylated proteins were precipitated and immunoblotted for ST6Gal-I. Both membrane and cleaved soluble forms of ST6Gal-I were transferred to SW948 and SW48 cells, as denoted by arrows. m, membrane; s, soluble. (D) SNA recognizes α2,6-sialylated ST6Gal-I. To verify that SNA pull-down experiments precipitated the sialylated ST6Gal-I form, pull-downs were conducted with parental SW48 cells that lack endogenous ST6Gal-I or SW48 cells stably expressing ST6Gal-I. Pull-downs were also conducted with neuraminidase-treated lysates from ST6Gal-I-overexpressing SW48 cells. The SNA pull-downs, as well as whole-cell lysates, were immunoblotted for ST6Gal-I. (E) ST6Gal-I in exosomesand exomeres is functional in recipient cells. SW948 cells were treated with exosomesor exomeres isolated from DiFi cells or untreated control. At different time points, cells were stained with FITC-SNA and then assessed for total cell-surface levels of α2,6-sialylation by flow cytometry. Data are presented as mean ± SEM; n = 3; *p < 0.05, **p < 0.01. (F) SW948 cells were treated with either exosomes or exomeres. Lysates were incubated with agarose-conjugated SNA lectin. α2,6-Sialylated proteins were precipitated and blotted for β1-integrin. See also Figure S6 .

Article Snippet: Sialyltransferase activity present in the lysates was determined using the Sialyltransferase Activity Kit from R&D Systems (cat# EA002) according to the vendor protocol with a few modifications Briefly, CMP-Sialic Acid (Sigma, cat# C8271) was used at a final concentration of 2.5 mM, N -acetyl-D-lactosamine (Sigma, cat# A7791) was used at a final concentration of 2.5 mM, the coupling phosphatase was used at a final concentration of 10 ng/μL, and CMP was used at a final concentration of 0.1 mM.

Techniques: Activity Assay, Derivative Assay, Western Blot, Stable Transfection, Transduction, Control, Plasmid Preparation, Expressing, Construct, Membrane, Incubation, Functional Assay, Isolation, Staining, Flow Cytometry

Journal: Cell reports

Article Title: Transfer of Functional Cargo in Exomeres

doi: 10.1016/j.celrep.2019.01.009

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Sialyltransferase activity present in the lysates was determined using the Sialyltransferase Activity Kit from R&D Systems (cat# EA002) according to the vendor protocol with a few modifications Briefly, CMP-Sialic Acid (Sigma, cat# C8271) was used at a final concentration of 2.5 mM, N -acetyl-D-lactosamine (Sigma, cat# A7791) was used at a final concentration of 2.5 mM, the coupling phosphatase was used at a final concentration of 10 ng/μL, and CMP was used at a final concentration of 0.1 mM.

Techniques: Recombinant, Plasmid Preparation, Labeling, shRNA, cDNA Synthesis, Isolation, SYBR Green Assay, Activity Assay, Software