Anti flotillin 1

Flotillin-1 and Snail were inserted into the pLVX vector. The Flotillin-1 shRNA sequences were cloned into the pSIH vector. The sequences of shRNAs are shown in Table S1.
Antibodies used in this study: anti-Flotillin-1 (#18634; Cell Signaling Technology, Danvers, MA, USA), anti-Snail (#3879; Cell Signaling Technology, Danvers, MA, USA), anti-E-cadherin (#3195; Cell Signaling Technology, Danvers, MA, USA), anti-N-cadherin (#13116; Cell Signaling Technology, Danvers, MA, USA), anti-Vimentin (#5741; Cell Signaling Technology, Danvers, MA, USA), anti-β-actin (A5441; Sigma-Aldrich, USA), anti-Flag (#14793; Cell Signaling Technology, Danvers, MA, USA), anti-HA (sc-53516; Santa Cruz Biotechnology, Dallas, TX, USA), Goat anti-Rabbit IgG (AS014; ABclonal, Hubei, China), Goat anti-mouse IgG (AS003; ABclonal, Hubei, China).

Cells were lysed in a buffer containing Tris (20 mM, pH 7.4), NaCl (150 mM), EDTA (1 mM), EGTA (1 mM), Triton X-100 (1%), sodium pyrophosphate (25 mM), NaF (1 mM), β-glycerophosphate (1 mM), sodium orthovanadate (0.1 mM), PMSF (1 mM), leupeptin (2 μg/mL) and aprotinin (10 μg/mL). An amount of 50 μg of total cell lysate protein was separated on SDS-PAGE gel, and transferred onto Hybond ECL nitrocellulose membranes (Amersham, UK), which were blocked with 5% skim milk at room temperature (RT) for 1 h, and incubated with individual primary (overnight at 4 °C) and secondary antibodies for 1 h at RT. Signals were subsequently developed using an ECL kit (Amersham, UK). Primary antibodies used were anti-γH2AX (1:100, Cell Signaling); anti-H2AX (1:1000, Millpore, Billerica, MA, USA); anti-phosphorylated DNAPK (1:1000, Abcam, Toronto, ON, Canada); anti-DNAPK (1:1000, Abcam); anti-phosph-CHK1 (S345) (1:500, Cell Signaling); anti-CHK1 (1:1000, Cell Signaling); anti-phospho-CHK2 (T68) (1:1000, Cell Signaling); anti-CHK2 (1:1000, Cell Signaling); anti-Flotillin-1 (1:1000, Cell Signaling); and anti-actin (1:1000, Santa Cruz, Dallas, TX, USA).

Typical exosome markers were identified in the EV fraction by Western blot according to previously published methods. In brief, 10 μg of EVs proteins was separated on a 10% polyacrylamide gel. Separated proteins were transferred to polyvinylidene difluoride membrane (PVDF; Thermo Scientific) in transfer buffer for 1 h at 100 V. The membrane was washed in wash buffer (PBS TWEEN 20 (0.1%) three times for 10 min and blocked with 5% skimmed milk in PBS TWEEN 20 (0.1%) for 1 h at room temperature under agitation. The blocked membrane was probed for previously identified exosome-specific markers using the following primary antibodies: rabbit polyclonal anti-CD63 (1:1000, sc-15363, Santa Cruz Biotechnology), mouse monoclonal anti-Alix (1:1000, sc-53540, Santa Cruz Biotechnology), and rabbit polyclonal anti-Flotillin-1 (1:1000, 3253, Cell Signaling), diluted in 5% skim milk in PBS TWEEN 20 (0.1%) at 4 °C overnight on the laboratory rocker. After overnight incubation, the membrane was washed 3 times for 10 min in wash buffer. Bound antibodies were detected using horseradish peroxidase linked anti-rabbit or anti-mouse secondary antibodies conjugates (KPL, Gaithersburg) and visualized using an ECL detecting system (Thermo Scientific).

The primary antibodies used in this study were as follows: anti-CD63 (dilution 1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-CD81 (dilution 1:1000, MBL, Nagoya, Japan), anti-TSG101 (dilution 1:1000, Abcam, Cambridge, MA), anti-flotillin-1 (dilution 1:1000, Cell Signaling Technology, Danvers, MA), anti-actinin-4 (dilution 1:1000, Cell Signaling Technology), anti-β-actin (dilution 1:5000, Sigma-Aldrich), anti-CLDN11 (dilution 1:1000, R&D Systems, Minneapolis, MN), anti-vimentin (dilution 1:1000 in western blotting and 1:100 in immunocytochemistry, Cell Signaling Technology), anti-ZO-1 (dilution 1:1000, Cell Signaling Technology), and anti-Snail (dilution 1:1000, Cell Signaling Technology). The secondary antibodies used in this study were as follows: horseradish peroxidase (HRP)-conjugated horse anti-mouse IgG (dilution 1:1000, Cell Signaling Technology), HRP-conjugated goat anti-rabbit IgG (dilution 1:1000, Cell Signaling Technology), and Alexa Fluor 488-conjugated goat anti-rabbit IgG (dilution 1:1000, Thermo Fisher Scientific).