*4.6. Immunogold*

For immuno-transmission electron microscopy (immuno-TEM), EVs were fixed in 4% formaldehyde and 0.1% glutaraldehyde for 15 min then dropped directly onto formvar/carbon coated grids and left for 20 min at room temperature. Grids were washed with PBS, blocked for 10 min with 0.5% bovine serum albumin in PBS, incubated for 20 min with mouse anti-CD63 primary antibody, incubated 20 min with rabbit anti-mouse secondary antibody and, finally, incubated with gold-labelled protein A for 10 min. Between each antibody incubation, grids were washed twice with PBS. Two washes, one in PBS and one in water, were performed before grids stained/embedded with 0.4% uranyl acetate/1.8% methyl cellulose for 10 min at 4 ◦C. Grids were allowed to dry at least for 20 min and samples were observed on a JEOL-JEM 1230 at 80 kV and images were recorded with a Morada digital camera.

#### *4.7. Cells and Vesicles Preparation for Fatty Acid Analysis*

For fatty acids analysis, about 3.6 × 106 of H-RasV12 expressing cells or control cells were washed twice with PBS, pelleted and stored at −80 ◦C. Total cellular lipids were extracted from 6 cell pellets from 3 different preparations and protein concentration was determined in each sample to normalize lipid content. For EVs, they were obtained from cell culture medium of the same preparations used for cell lipid extraction, pooled, and 8 μg of proteins used for each analysis. Lipid extraction was carried out as previously reported [50,51]. Extracts were dried under nitrogen and resuspended in methanol prior to be submitted for analyses. An acid hydrolysis was carried out by dissolving the methanol fraction in chloroform/methanol 1:1 (*v*/*v*). Then, 1 M HCl:methanol (1:1, *v*/*v*) solution was added to the total lipid extract and the mixed solution shaken for 2 h. Chloroform:water (1:1 *v*/*v*) was added and the organic phase collected and dried under nitrogen flow. The residue was dissolved in methanol. For fatty acid quantification, the MS analysis was carried out with a selective ion monitoring-tandem mass spectrometry (SIM-MS/MS) method. Quantitative analysis was performed with calibration curves. An ESI source connected with an API 4000 triple quadrupole instrument (AB Sciex, Old Connecticut Path, Framingham, MA USA) was used. The mobile phases were: Water/10 mM isopropylethylamine/15 mM acetic acid (phase A) and methanol (phase B). MultiQuant™ software version 3.0.2 (AB Sciex, Old Connecticut Path, Framingham, MA, USA) was used for data analysis and peak review of chromatograms. Quantitative evaluation of phospholipid families was performed based on standard curves. Quantitative data were normalized on the protein content of cells or vesicles. An external standard for each phospholipid family was used for the semi-quantitative analysis. Data were normalized on protein content.

#### *4.8. qRT-PCR*

RNA was extracted and retro-transcribed as previously described [12]. cDNA was used to determine the expression of genes listed in Supplementary Table S1. cDNA was used to determine transcript levels by qRT-PCR in a StepOne RT-PCR machine (Applied Biosystems, Foster City, CA, USA) using SYBR® Select Master Mix (Life Technologies, Carlsbad, CA, USA). Reactions were performed in triplicate and GAPDH used as endogenous control. Data were analysed using the ΔΔ*C*t method. Δ*C*t was calculated subtracting the average *C*t value of *GAPDH* to the average *C*t value of a specific gene for each sample, then ΔΔ*C*t as the difference between the Δ*C*t for each sample and the Δ*C*t of empty vector transfected fibroblasts as control. The reported fold expression, expressed as Relative Quantity, was calculated by 2−ΔΔ*C*<sup>t</sup> .
