Gm130

Both types of cells were lysed on ice with M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific, USA) supplemented with 10% protease inhibitor cocktail (Roche, Mannheim, Germany) to isolate total protein and then measured by a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, USA) in accordance with the manufacturer’s instructions. Protein samples supplemented with loading buffer in equal proportions were electrophoresed on NuPAGE™ Bis-Tris Protein Gels (Invitrogen, Waltham, MA, USA) and then transferred onto polyvinylidene fluoride (PVDF) membranes. After incubation with 5% skim milk (5% w/v) for 2 h at room temperature, the membranes were co-incubated overnight at 4 °C with the following primary antibodies specific for GAPDH (1:1000; Cell Signaling Technology, USA), Runx2 (1:1000; Cell Signaling Technology, Danvers, MA, USA), Osx (1:1000; Abcam, Cambridge, UK), Ocn (1:2000; Abcam, UK), ELK4 (1:1000; Proteintech, Rosemont, IL, USA), GM130 (1:1000; Proteintech, USA), CD9 (1:1000; Proteintech, USA), and TSG101 (1:1000; Proteintech, USA). Next, the peroxidase-conjugated secondary antibody (1:5000; ZS-GB-BIO, Beijing, China) was utilized to treat the PVDF membranes for 2 h at room temperature. The ECL kit (Thermo Fisher Scientific, USA) was employed to visualize the signals. Densitometry analysis was conducted with ImageJ software.

Extracellular vesicles were examined by transmission electron microscopy (TEM, Hitachi, Japan) and dynamic light scattering (DLS, Malvern Instruments Ltd., Worcestershire, UK). Then western blot was carried out to identify the ADSCs-EV by several surface markers. Briefly, equal amounts of total protein samples from ADSCs-EV (30 µg) were loaded in each well of SDS-PAGE gels. The gels were subsequently transferred onto PVDF membranes and incubated overnight at 4 °C with following primary antibodys: CD9 (Cat #A1703; ABclonal), GM130 (Cat #11308‐1‐AP; Proteintech), CD63 (Cat #ab134045; Abcam) and β‐tublin (Cat #10094‐1‐AP; Proteintech). On the following day, the membranes were incubated with HRP-conjugated antibody (Aspen, China) for 1 h. After incubation, the membranes were again washed 3 times with PBS and exposed to X-ray films using ECL detection reagents (#WP20005, Thermo Fisher). Changes in hydrodynamic diameters were monitored for 8 d by DLS to test the stability of the ADSCs-EV in vitro. A Cell Counting Kit-8 (CCK8) kit (SAB, College Park, MD, USA) was used to identify the cytotoxicity of different concentrations of ADSCs-EV and ⁶⁸ Ga-L-NETA-DBCO in HCT116 human colon cancer cells.