Hi Ilaria Salvato . For a quick check, you could do a slot blot of media from treated and non-treated cells and stain for common epithelial membrane proteins.

https://www.merckmillipore.com/INTL/en/product/Epithelial-Cell-Markers,MM_NF-C130854

The best option would do to do SEC on the supernatant of samples before and after treatment, then stain for CD9/CD63/CD81 and see by EM if the vesicles you have in one condition are absent in the other. That's what's best. A dirtier option is do ultracentrifugation of the same supernatants and check by western blot whether you can detect these markers.

On a side note, GW4869 does not work on all cell lines, so don't asume it does.

You might need to check whether your GW4869 is working by an independent assay such as protection from cell death. As GW4869 is a noncompetitive neutral sphingomyelinase (N-SMase) inhibitor, which inhibits  initial accumulation of ceramide, it reduces EV secretion by blocking ceramide-dependent

budding of intraluminal vesicles (ILV) into the lumen of multivesicular bodies .

Due to its effect on ceramide, it can protect from TNF-induced cell death,  in a dose-dependent manner, as can be monitored by  nuclear condensation, caspase activation, PARP cleavage, and trypan blue uptake.

See: DOI: 10.1074/jbc.M206747200