15 cm dishes

A431 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM)
(A.T.C.C., VA) supplemented with 10% FBS (Atlas Biologicals, CO), and
penicillin/streptomycin (ThermoFisher, MA). Trypsin/EDTA was
obtained from Gibco (ThermoFisher, MA). Unless otherwise noted, chemicals were
obtained from Sigma Chemical Co. (Sigma, MO). Recombinant EGF and c-Fos were
obtained from Active Motif (Active Motif, CA). Recombinant Ras was obtained from
Cytoskeleton, Inc. (Cytoskeleton, CO). Human EGF was obtained from Cytoskeleton,
Inc. (Cytoskeleton, CO). For EGF stimulation experiments, A431 cells were serum
restricted for 24 h with serum-free DMEM in order to synchronize the cells. The
cells were then treated with 33 ng/ml EGF for 0.5, 2, 5, 15, and 60 min
in individual 15-cm dishes (Corning, NY), followed by subsequent lysis with
BlastR lysis buffer (Cytoskeleton, CO).

Mouse muscle myoblast cells C2C12 were obtained from American Tissue and Cell Culture (ATCC, Manassas VA, USA) and maintained in DMEM supplemented with 20% FBS and penicillin/streptomycin at 37 °C and 5% CO2. For differentiation, the cells were grown to confluency in 15 cm dishes (Corning, NY), switched to DMEM with 2% FBS, and allowed to differentiate for five days. The proliferating C2C12 cells were labeled with SILAC amino acids. Briefly, the C2C12 cells were cultured in DMEM supplemented with 20% FBS and SILAC amino acids such as L-lysine (13C615N2) and L-arginine (13C615N4). After five doubling cycles, all the proliferating C2C12 cells were fully labeled. The SILAC-labelled proliferating C2C12 cells and the non-labelled differentiated C2C12 cells were prepared separately.

A431 human epidermoid carcinoma cells and 3T3-swiss mouse fibroblast cells were obtained from ATCC. Cells were grown in DMEM media (ATCC, Manassas, VA, USA) supplemented with 10% FBS (Atlas Biologicals, Fort Collins, CO, USA) and penicillin/streptomycin (ThermoFisher, Waltham, MA, USA). TrypLE Express was obtained from Gibco (ThermoFisher, Waltham, MA, USA). Unless otherwise noted, chemicals were obtained from Sigma Chemical Co. (Sigma, St. Louis, MO, USA). For all Western blot and IP analysis experiments, A431 cells at 60% confluency, post 5 days of cell passage, were treated with 100 μM H₂O₂ for 2 h in individual 15 cm dishes (Corning, Corning, NY, USA).

MCF-7 (Bioresource Collection and Research Center, Hsinchu, Taiwan) cells were cultured and grown in 15 cm dishes (Corning). When cells reached approximately 80% confluence, cells were washed with phosphate buffered saline (PBS) (Gibco) and transferred to fresh media containing 1% EV-free serum (Gibco) for 24 h. Media was then collected and subjected to centrifugation at 500×g for 5 min to pellet cells, then 2000×g for 15 min to remove cellular debris (Eppendorf, Hamburg, Germany). The supernatant was then transferred to ultracentrifuge tubes (Beckman Coulter, Pasadena, CA, USA) and ultracentrifuged (Beckman Coulter) at 10,000×g for 30 min twice to pellet and remove large vesicles. The supernatant was then transferred to new ultracentrifuge tubes and ultracentrifuged twice at 100,000×g for 60 min to pellet EVs. EVs were resuspended in PBS and then stored at 4 °C. Protein content of EVs was determined by BCA (Thermo Fisher Scientific) and treatment dosing was determined by EV protein concentration. Particle size was measured using qNano Gold (Izon, Science Ltd., Burnside, New Zealand) by Tunable Resistive Pulse Sensing. Data was recorded and analyzed using Izon Control Suite Software (version 2.2.2.111). Particle size reported are representative of three separate measurements of EVs.