Fv1000 confocal laser scanning biological microscope

In situ proximity ligation assay was performed according to the instructions for the Duolink in situ PLA kit (Sigma-Aldrich). Anti-VDAC1, anti-IP3R1, or anti-GRP75 antibodies were used to visualize MAM formation. Fluorescent blobs were detected using a FV1000 confocal laser scanning biological microscope (Olympus, Tokyo, Japan) or an ImageXpress Micro Confocal Imaging System (Molecular Devices). The number of detected blobs per nucleus was calculated by ImageJ software or MetaMorph Microscopy Software (Molecular Devices).

For the in vivo tracking of sEVs, we isolated sEVs from the conditioned medium of young ADSCs and labeled them after the first ultracentrifuge with 5 μM PKH26 (Sigma-Aldrich, MINI26-1KT), after that, sEVs were resuspended in PBS and washed with another ultracentrifuge. Twenty micrograms of PKH26-labeled sEVs were injected in old mice (18 months), mice were euthanized 24 hours later, and kidney and gastrocnemius were obtained and fixed with 2% PFA for 24 hours. Forty-micrometer slices were mounted and stained with Hoechst (Invitrogen) 1:1000 for 30 min at RT. Coverslips were mounted with an aqueous mounting medium and sealed with nail polish. Images were acquired with Olympus FV1000 confocal laser scanning biological microscope. Images were processed in ImageJ, all levels were adjusted equally, and the ratios were not altered.

The level of phosphorylated histone variant H2AX (γ‐H2AX), which is a surrogate marker of DNA damage is detected using immunofluorescence assay. In brief, CaSki‐RR and SiHa‐RR cells with FBXO6 overexpression alone or together with CD147 overexpression were plated onto culture slides (Corning Inc, Corning, NY, USA). When cells reached ~50% of confluence, cells were subjected to 17‐AAG (20 nM/24 h for CaSki‐RR and 50 nM/24 h for SiHa‐RR) or control pre‐treatment and then treated with 2 Gy irradiation. At 6 h later, the cells were fixed with buffered formaldehyde for 15 min and then permeabilized with 0.02% Triton. The cells were then blocked in 3% albumin in Dulbecco's phosphate‐buffered saline (DPBS) for 1 h. Then, the slides were incubated with a primary antibody against γ‐H2AX (AP0687, ABclonal, Wuhan, China) in a 1:200 dilution in the 3% albumin solution overnight at 4°C. Then, the slides were thoroughly washed and incubated with goat anti‐rabbit IgG (H&L) (Alexa Fluor 647) (ab150079, Abcam, Cambridge, UK) secondary antibody in a 1:500 concentration in albumin. The slides were kept in darkness for 1 h before washing. The slides were mounted using VECTASHIELD Antifade Mounting Medium with 4', 6‐diamidino‐2‐phenylindole (DAPI). Then, fluorescence images were captured using an FV1000 confocal laser scanning biological microscope (Olympus, Tokyo, Japan).