Triton 100

The cell supernatant was centrifuged at 500 g × 10 min and 2,000 g × 20 min to remove the floating cells and cell debris, and then centrifuged at 20,000 g × 30 min at 4°C to remove the precipitate and collect the supernatant. After centrifugation at 100,000 g × 70 min, the precipitate was the EVs (Wang et al., 2018 (link)). The EVs precipitate was washed with PBS, centrifuged again at 100,000 g × 70 min, and suspended in 1 mL PBS. The suspension was placed on the top of 30% sucrose cushions to form a clear interface layer, and centrifuged at 100,000 g × 70 min at 4°C. The PBS layer was discarded, and the deuteroxide layer was retained. The deuteroxide was mixed with at least five times the volume of PBS, and centrifuged again at 100,000 g × 70 min to obtain the purified EVs. The EVs were filtered and sterilized with 0.22 μm membrane, and then put into aseptic Ependorf tube. The EVs were grouped and frozen at −80°C. The EVs of some groups were incubated with 1 μg/mL ribonuclease A (Sigma-Aldrich) at 37°C for 30 min or treated with 0.1% Triton × 100 (Beyotime, Shanghai, China) for 30 min (Wang et al., 2018 (link)) for subsequent analysis.

Based on a previous study [33 (link)], CAFs-EVs were treated with 1 μg/mL RNase A (Invitrogen) or RNase A in combination with 0.1% Triton × 100 (Beyotime) at normal temperature. Subsequently, the expression patterns of lncRNA SNHG12 were detected by quantitative real-time polymerase chain reaction (qRT-PCR). The experiment was repeated 3 times independently, with at least 3 duplicate wells set for each group of cells in each experiment.

Cultured cells were rinsed with PBS and then fixed with 4% PFA for 20 min. Cells were treated for 5–10 min with 0.5% Triton-100 (Beyotime; cat. no. P0096) and then 4% BSA was added to reduce non-specific staining. Specimens were incubated with primary antibodies (S Table 1) at 37 °C overnight, followed by incubation with secondary antibodies (S Table 1). For the negative controls, the primary antibodies were replaced with PBS and cells were only incubated with the secondary antibodies. The specimens were analyzed and photographed using a Zeiss LSM 780 confocal microscope (Zeiss, Jena, Germany).