Journal: Journal of Extracellular Vesicles
Article Title: The αvβ6 integrin in cancer cell-derived small extracellular vesicles enhances angiogenesis
Figure Lengend Snippet: Transfer of prostate cancer cell-derived αvβ6-positive sEVs to microvascular endothelial cells regulates survivin levels. (a) HMEC1 were plated (2 × 10 5 ) in six-well plates and incubated with PBS or sEVs derived from PC3-shCtrl, -shβ6 or -shβ5 cells for 18 h and after incubation, the TCL were analysed by IB under reducing conditions for expression of pSTAT1(Y701), STAT1 and ACTIN (loading control). (b) HMEC1 were plated (2 × 10 5 ) in six-well plates and incubated with PBS or sEVs derived from PC3-shCtrl, -shβ6 or -shβ5 cells for 18 h and after incubation, the TCL were analysed by IB under reducing conditions for expression of survivin and ACTIN (loading control). (c) Left panel, IB analysis for expression of β6 integrin subunit, CD63, CD81 and CANX (non-reducing conditions); right panel, IB analysis for expression of β5 integrin subunit, ALIX, TSG101, CD9, survivin and CANX (reducing conditions) in TCL and sEV lysates from PC3 cells. (d) Iodixanol density gradient analysis of PC3 sEVs was performed as described in the Materials and methods. Expression of ALIX, TSG101, survivin, CANX (reducing conditions), CD63 and CD81 (non-reducing conditions) analysed by IB of 10 consecutive iodixanol density gradient fractions is shown. (e) IB analysis for expression of ALIX and survivin (reducing conditions) in TCL and sEV lysates from PC3-shCtrl and -shβ6 cells. Different gels were used to separate samples under reducing or non- reducing conditions.
Article Snippet: Briefly, the LEVs obtained from PC3-parental cells or sEVs obtained from PC3 cells (PC3-parental, -shCtrl, -shβ5 and -shβ6) or C4-2B cells (C4-2B-Mock, -αvβ6) were suspended in 1.636 mL of 30% iodixanol solution [made by mixing 1:1 of 60% (wt/vol) stock solution of iodixanol (OptiPrep™, Sigma # 1556) with a buffer (0.25 M sucrose, 10 mM Tris pH 8.0, 1 mM EDTA, pH 7.4)] and layered at the bottom of an ultracentrifugation tube.
Techniques: Derivative Assay, Incubation, Expressing