Journal: Journal of Extracellular Vesicles
Article Title: Cholesterol Deficiency Directs Autophagy‐Dependent Secretion of Extracellular Vesicles
doi: 10.1002/jev2.70218
Figure Lengend Snippet: sEVs from cholesterol‐depleted cells are more readily internalized. (A) Stably transfected HEK293T cells overexpressing mEmerald‐CD9 or eGFP‐CD63. Scale bar, 200 µm. (B) Experimental design for isolation of mEmerald‐CD9 and eGFP‐CD63 sEVs and quantitation of uptake by recipient cells. (C) Representative 3D reconstructions of target celluptake of mEmerald‐CD9 sEVs. Plasma membrane visualized with concanavalin A (ConA). Scale bar, 25 µm. (D) Representative images of target cells incubated with sEVs from cells treated with vehicle, simvastatin, or AY9944. Scale bar, 25 µm. (E) Quantified uptake in recipient cells exposed to 1 × 10 8 or 2.5 × 10 8 mEmerald‐CD9 sEVs (mean ± SEM; n = 21, 7 images analyzed from 3 independent experiments). Two‐way ANOVA (Interaction effect: F2,120 = 26.31, p < 0.0001; Treatment effect: F2,120 = 70.3, p < 0.0001; Dose effect: F1,120 = 152.1, p < 0.0001). Sidak's multiple comparisons test (** p ≤ 0.005, **** p < 0.0001). (F) Quantified uptake in recipient cells exposed to 1 × 10 8 or 2.5 × 10 8 eGFP‐CD63 sEVs (mean ± SEM; n = 12, 4 images analyzed from 3 independent experiments). Two‐way ANOVA (interaction effect: F2,66 = 5.633, p ≤ 0.0055; Treatment effect: F2,66 = 40.73, p < 0.0001; Dose effect: F1,66 = 58.16, p < 0.0001). Sidak's multiple comparisons test (* p ≤ 0.05, ** p ≤ 0.005, **** p < 0.0001).
Article Snippet: Plasmids housing genes for proteins involved in sEV biogenesis or mature autophagosome formation fused to fluorescent reporters, including mEmerald‐CD9 (a gift from Michael Davidson, Addgene plasmid # 54029), eGFP‐CD63 (a gift from Paul Luzio, Addgene plasmid # 62964) and mCherry‐LC3B (a gift from David Rubinsztein, Addgene plasmid # 40827), were isolated and purified (GeneJET Plasmid Maxiprep Kit, Cat. # K0491).
Techniques: Stable Transfection, Transfection, Isolation, Quantitation Assay, Clinical Proteomics, Membrane, Incubation
Addgene inc is a verified supplier 
