Ripa buffer

Proteins were extracted and quantified using radio immunoprecipitation assay (RIPA) buffer (Biovision, American) and a protein concentration detection kit (absin, Shanghai, China). The prepared protein was separated by polyacrylamide-sodium dodecyl sulfate (SDS) gels and then transferred onto polyvinylidene fluoride (PVDF) membranes (Roche, Switzerland). Next, the membranes were incubated overnight at 4 ℃ with polyclonal antibodies: cleaved-caspase-1 (#4199T, 1:1,000, Cell Signaling Technology, USA), NOD-like receptor family pyrin domain containing 3 (NLRP3) (ab263899, 1:1,000, Abcam, UK), apoptosis-associated speck-like protein containing a CARD (ASC) (#13833S, 1:1,000, Cell Signaling Technology, USA), gasdermin D (GSDMD)-N (ab215203, 1:1,000, Abcam, UK), interleukin (IL)-1β (ab216995, 1:1,000, Abcam, UK), IL-18 (ab243091, 1:1,000, Abcam, UK), and β-actin (ab8226, 1:1,000, Abcam, UK). After washing with Tris-HCl buffered saline with 0.1% (v/v) Tween 20 (TBST), the membranes were incubated with a secondary antibody, and protein expression intensity was determined by enhanced chemiluminescent (ECL) kit (Beyotime, Beijing, China).

Collected cells were lysed with RIPA buffer (BioVision, Inc.) and the supernatant was extracted for SDS-PAGE analysis. After quantification using the Bradford assay, protein lysates (10 µg/lane) were separated by 8% SDS-PAGE, transferred to PVDF membranes and blocked with TBS containing 5% non-fat milk at room temperature for 1 h. The membranes were probed with mouse monoclonal anti-Flag antibody (1:3,000; cat. no. 81069; ProteinTech Group, Inc.) or PTPRA antibody (1:2,000; cat. no. 13079–1-AP; ProteinTech Group, Inc.), β-actin antibody (1:2,000; cat. no. Ag27042; ProteinTech Group, Inc.) for 1–2 h at room temperature. The blots were then incubated and reprobed with horseradish peroxidase-conjugated secondary antibody (1:2,000; cat. no. SA00001-1; ProteinTech Group, Inc.) for 1 h at room temperature. Band signals were visualized using an ECL system (GE Healthcare). The following antibodies were diluted in TBS and used for immunoblotting: Anti-FLAG, anti-β-actin and anti-PTPRA.

To validate our findings on crude cell-free RNA in EV samples with higher purity, we performed additional experiments using a subgroup of the initial patient population (n = 14). In these experiments, crude EVs were enriched from 1 ml arterial or venous serum using precipitation as described above and further purified by size-exclusion chromatography (SEC, qEVoriginal, Izon Bioscience, Cambridge, UK). Crude EV pellets were resuspended in 500 µl Dulbecco’s Phosphate Buffered Saline (DPBS) and loaded onto qEV columns as per the manufacturer’s instructions. The EV-containing fractions 7–9 were collected, pooled and pelleted by ultracentrifugation for 2 h at 200,000 x g (Beckman Optima LE-80K Ultracentrifuge, SW60 Rotor, k-factor 84.0, Brea, USA). Total RNA was subsequently extracted from pellets using the miRNeasy Micro Kit (Qiagen, Venlo, The Netherlands). Preparation of sequencing libraries, sequencing-by-synthesis and differential gene expression (DGE) analyses were performed as described below. For EV characterisation experiments, pellets were resuspended in 30 µl sterile-filtered DPBS or lysed in 1x RIPA buffer (Abcam, Cambridge, UK).