Polyvinylidene di uoride membrane

Total protein was isolated with RIPA buffer (Beyotime) and determined with a BCA protein assay kit (Beyotime). Then the extracts were separated by sodium dodecyl sulfonate-polyacrylamide gel (Solarbio) and transferred to polyvinylidene di uoride membranes (Millipore, Billerica, MA, USA). Next, the membranes were maintained in 5% slim milk for 1 h and incubated overnight at 4 °C with primary antibodies: Ki67 (ab16667;
Abcam), Twist1 (ab49254; Abcam), E-cadherin (ab231303; Abcam), LIPH (ab192615; Abcam), GALNT7
(ab113743; Abcam), PSD3 (ab62194; Abcam), ITGA2 (ab133557; Abcam) or GAPDH (ab181602; Abcam).
Afterward, the samples were subjected to secondary antibody (ab205719; Abcam) for 2 h at room temperature. At last, the protein bands were determined using ECL western blot kit (Beyotime).

The expression of caspase-3, cleaved caspase-3, E-cadherin, N-cadherin, Vimentin, Bax, Bcl-2 and btubulin in samples and in cells irradiated by 6 MV x-ray in different groups were detected by WB. Total proteins from cells were centrifuged for 20 min at 13,000´g. Proteins were then shifted to polyvinylidene di uoride membranes (Millipore, Shanghai, China), closed with 5% non-fat milk in tris-buffered saline with Tween (TBST, 20 mM Tris-HCl, 150 mM NaCl, 0.05% Tween 20, pH 8.0) and buffered for 1 h. The target proteins were detected with different antibodies at 4°C overnight, and washed 4 times with TBST for 30 min. Then, the secondary antibodies were bioconjugated with horseradish peroxidase at a dilution (1:10000) for 1 h at room temperature and washed 4 times with TBST. The β-tubulin was used as the internal reference. The enhanced chemiluminescence (ECL) method was applied for development, and cassette exposure was used for development and xation. Bands were visualised using a UVP detection system and band intensity that was quanti ed using LAB WORKS 4.6. All experimental results were repeated 3 times.

Cell pellets or puri ed exosomes were harvested and lysed with RIPA lysis buffer (Beyotime) supplemented with the protease inhibitor phenylmethylsulfonyl uoride (PMSF) (1 mM; Beyotime) and a phosphatase inhibitor (Beyotime). Cell lysates and exosomes were heat denatured at 100℃ incubator for 10 min, separated by 10% or 8% SDS-PAGE and transferred to polyvinylidene di uoride membranes (Millipore). The proteins were identi ed by incubation with the appropriate primary antibody and then with an HRP-conjugated secondary antibody (Pierce). Immunoreactions were visualized with ECL reagent (Pierce), and images were acquired using a ChemiDoc Touch Imaging System (Bio-Rad) and analyzed with Image Lab software. The densities of the corresponding bands were quanti ed using ImageJ software.
Cell viability assay SK-OV-3, HCT116, HEp-2 and MDA-MB-231 cells were seeded into 96-well plates at a density of 1 × 10 4 cells per well one day before exposure to exosomes. Cells in triplicate wells were mock treated (Mock) or incubated with 1 µg of puri ed exosomes produced by HEK-293 cells transfected with plasmids encoding either miR-HER2-E1 or the nontargeting (NT) miRNA. After 72 h of incubation, the relative cell viability was determined by a CCK8 assay according to the manufacturer's protocol.