Flotillin 1

PKH67 was purchased from Sigma, MitoTracker deep red and CellLight™ Mitochondria-RFP, BacMam 2.0 were obtained from ThermoFisher. Growth Factor Reduced Matrigel was obtained from BD Biosciences (BD No. 354230). Antibodies to Ser616-phosphohry lated Drp1, Drp1, MFN1, MFN2, Ser473-phosphorylated AKT, Thr308-phosphorylated Akt, Akt, EGFR, MMP9, ILK, HIF1α, Calnexin, VDAC, Flotillin 1, E-cadherin, Vimentin, SNAIL, Ki67, cleaved-Caspase 3, phosphorylated Ezrin-Radixin-Moesin (pERM) complex were obtained from Cell Signaling. An antibody to hDlg was from Santa Cruz. An antibody to Tsg101 was from ProteinTech. Antibodies to CD63 and CD9 was from Abcam. An antibody to β-actin was from Sigma. The Akt inhibitor MK2206 (1 μM) was purchased from Selleck Chemicals and ILK inhibitor Cpd22 (600 nM) was purchased from Sigma.

Culture supernatants and cell lysates were analyzed by western blotting. Proteins in these samples were separated by SDS-PAGE (under reducing and non-reducing conditions) and then transferred to polyvinylidene difluoride membranes. Antibodies to the following proteins were used as primary antibody to detect them: HRF (anti-TPT1 mAb clone 2C4, Catalog #, H00007178-M03) from Abnova was used unless otherwise depicted), Flotillin-1, CD9, Annexin, and CD54 (Cell Signaling Technology). Horseradish peroxidase-linked anti-mouse IgG Ab and Horseradish peroxidase-linked anti-rabbit IgG Ab were used as secondary antibody.

Exosome purity and protein contamination were determined by Western blot for exosome-specific proteins Flotillin-1 and CD9 and plasma protein stain Ponceau S. Briefly, Western blots were conducted by taking an aliquot of fractions 4 to 12 of the final density gradient; 37.5 μL of each fraction was mixed with 4 × Laemmli buffer containing 2-mercaptoethanol. The sample was boiled at 95°C for 5 minutes and resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on a 10% gel. The gel was transferred onto a polyvinylidene difluoride membrane using a standard tank protocol. The membrane was stained with 0.1% w/v Ponceau S to visualize total protein, followed by a 1-hour block in 5% nonfat milk. The membrane was probed with CD9 (Abcam, Cambridge, Mass) and Flotillin-1 (Cell Signaling Technology, Danvers, Mass) in 1% nonfat milk. The primary antibodies were used at a dilution of 1:500. The membrane was washed with 0.1% PBS-T detergent and probed with an anti-rabbit horseradish peroxidase antibody (1:1000 dilution; ThermoFisher Scientific, Waltham, Mass). Subsequently, the membrane was rewashed with 0.1% PBS-T and visualized using enhanced chemiluminescence on an ImageQuant LAS 4000 (GE Healthcare Life Sciences, Marlborough, Mass).