Odyssey v3

Cells were lysed in the presence of protease and phosphatase inhibitors (Roche Diagnostics, Indianapolis, IN, USA). Whole-cell lysates were subjected to SDS-polyacrylamide gel electrophoresis, electrophoretically transferred onto polyvinylidene difluoride (PVDF) membranes (Thermo Fisher Inc., Rockford, IL, USA) and immunoblotted with anti-Bcl-2 (ab692, Abcam, Cambridge, MA, USA), -Bcl-xL (2764), -Mcl-1 (4572), -PARP (9542), -Bim (2819), -γH2AX (2577), -Bak (3814), -Bax (2774), -cleaved caspase-3 (9661, designated -cf caspase-3, Cell Signaling Technology, Danvers, MA, USA) or -β-actin (A2228, Sigma-Aldrich) antibody, as previously described.^{22 (link),23 (link)} Immunoreactive proteins were visualized using the Odyssey Infrared Imaging System (Li-Cor, Lincoln, NE, USA), as described by the manufacturer. Western blots were repeated at least three times and one representative blot is shown. Densitometry measurements were made using Odyssey V3.0 (Li-Cor), normalized to β-actin, and calculated as the fold-change compared to the corresponding no drug treatment control.

Protein isolation and Western blot were carried out in the same manner as previously described [46 (link),47 (link),48 (link)]. Proteins from EVs were extracted with RIPA buffer (10 mM Tris, 150 nM NaCl, 1% deoxycholic acid, 1% Triton X, 0.1% SDS, and 1 mM EDTA) containing protease inhibitors (aprotinin, PMSF, and sodium orthovanadate). A Pierce^TM bicinchoninic acid (BCA) protein assay kit was used to assess protein content (Thermo Fisher Scientific, Waltham, MA, USA). A 4–20% Tris-Glycine gel (Bio-Rad, Hercules, CA, USA) was loaded with equal quantities of protein (20 g/lane) and electrophoresed under nonreducing conditions. Proteins were transferred to a PVDF membrane, blocked for 2 h in blocking buffer (PBS, 0.05 percent Tween20^®, 5% milk), and then probed overnight with the following primary antibodies: ALIX (Sc-166952; Santa Cruz, CA, USA); CD81 (Sc-7637; Santa Cruz); and GM130 (#12480; Cell Signaling, Danvers, MA, USA). The membranes were rinsed the next day and treated for 1 h at room temperature (RT) with the secondary antibodies donkey anti-mouse IRDye 800CW and donkey anti-rabbit IRDye 680RD (1:2000, LI-COR Biosciences, Lincoln, NE, USA). All antibodies were diluted according to the manufacturer’s instructions. A fluorescence scanner was used to photograph the membranes (Odyssey v.3.0, LI-COR Biosciences).

Cells were lysed in the presence of protease and phosphatase inhibitors (Roche Diagnostics, Indianapolis, IN, USA). Whole-cell lysates were subjected to SDS-polyacrylamide gel electrophoresis, electrophoretically transferred onto polyvinylidene difluoride (PVDF) membranes (Thermo Fisher, Inc., Rockford, IL, USA), and immunoblotted with anti-Mcl-1 (4572), -PARP (9542), -Bim (2819), -Bak (3814), -Bax (2774), -c-Myc (5605s), -cleaved caspase-3 (9661, designated -cf-Cas3; Cell Signaling Technology, Danvers, MA, USA), or -β-actin (A2228; Sigma-Aldrich) antibody, as previously described.^{37 (link),38 (link)} Immunoreactive proteins were visualized using the Odyssey Infrared Imaging System (Li-Cor, Lincoln, NE, USA), as described by the manufacturer. Western blots were repeated at least three times, and one representative blot is shown. Densitometry measurements were made using Odyssey V3.0 (Li-Cor), normalized to β-actin, and calculated as the fold change compared to the corresponding no drug treatment control.