Confocal uorescence microscope

A total of 5 × 10 4 cells were seeded into a confocal laser dish one day before 8 Gy irradiation. Four hours post-irradiation, cells were xed in 4% paraformaldehyde at room temperature for 30 minutes and permeabilized in 0.1% Triton X-100 for 2 hours. Cells were then blocked with 5% BSA for 90 minutes and washed with PBS. After incubation with the primary antibody γ-H2AX (1:400; Cell Signalling Technology) overnight at 4℃, the cells were washed with PBS and incubated with an Alexa Fluor 555-conjugated secondary antibody (Beyotime) for 90 minutes. Cells were washed with PBS and treated with DAPI staining solution for 20 minutes and then observed using a confocal uorescence microscope (Leica).

Following treatment with 2 µg/mL MIF in the presence or absence of 50 µM 4-IPP for 24 h, primary joint capsule broblasts were xed in 4% paraformaldehyde with 0.1% Triton X-100 (Sigma, St Louis, MO, USA)
and subsequently incubated with 4% goat serum to block nonspeci c binding. Then, cells were incubated with anti-TGF-β1 (Servicebio, Wuhan, China, 1:100) primary antibodies overnight. Fluorescent secondary antibodies of FITC-labeled goat anti-rabbit IgG (Sigma, 1:400) were used to visualize the corresponding subsets. Cells were then stained with 4,6-diamidino-2-phenylindole (DAPI; Sigma, 1:4000) and phalloidin (Abcam, 1:1000), followed by observation under a confocal uorescence microscope (Leica, Germany).

FBS was depleted of EVs by ultracentrifugation at 140,000 g and 4 °C for 16 hours, and the supernatant was collected and ltered using a 0.22 µm lter (Millipore, USA). EVs derived from blood samples and cell medium were isolated by differential centrifugation as previously described (15) . Before EV isolation, cells were cultured in normal medium until 50% con uency and then washed with phosphate-buffered saline (PBS) three times; and the medium was replaced with RPMI-1640 with 10% EV-depleted FBS and cultured under normoxic or hypoxic conditions. After 48 hours, the cell culture medium was harvested (50 ml), and EVs were isolated by differential centrifugation as previously described. The EVs were used immediately for further experiments. The size distribution and concentration of EVs were analysed by nanoparticle tracking analysis (NTA) using a ZetaView particle tracker from ParticleMetrix (Meerbusch, Germany). We used a transmission electron microscope (TEM; JEM-1200EX, JEOL Ltd., Japan) to observe the structure of EVs. CD63, CD81 and Alix were used as exosomal markers, and calnexin was used as a negative control for EVs. PKH67 (Sigma-Aldrich, USA) was used to label EVs. Twenty-four hours after PKH67labelled EVs were incubated with OSCC cells, DAPI was used for nuclei staining. The cells were visualized with a confocal uorescence microscope (Leica, Germany).