Fig. 5.
Modulation of actin cytoskeleton mediated by Gβγ subunit signaling coupled with S1PRs plays a key role in the formation of exosome-like vesicles. (A–D) HeLa cells pretreated with PTX (200 ng/mL) for 2 h (A), YM254890 (20 μM) for 1 h (B), Y27632 (20 μM) for 1 h (C), or gallein (20 μM) for 1 h (D) were incubated with solvent or staurosporine (1 μM) for 16 h. Exosomal fractions were Western blotted for CD63, S1PR1, or S1PR3 (Upper), and protein concentrations were measured by Bradford assay (Lower). (E) eGFP-CD63 HeLa cells treated with staurosporine were stained with phalloidin. Confocal microscopic images were taken. (Magnification: 400×; Insets: 1,000×.) The arrowheads designate CD63+ endosomes enclosed by F-actin. (F) HeLa cells preincubated with cytochalasin D (20 μM) or solvent were treated with staurosporine for 16 h, and exosomal fractions were prepared. CD63, S1PR1, and S1PR3 were detected by Western blot (Upper), and protein concentrations in the resuspended fractions were measured (Lower). Data in duplicate are presented as mean ± SD; *P < 0.001 versus control in A, D, and F).