Protease inhibitor cocktail

To remove dead cells and debris, the conditioned medium was filtered through a 0.45 µm syringe filter (2,542,903, PerkinElmer, Waltham, MA) and stored at − 20 °C. For intracellular (IC) protein analysis, cells were lysed for 30 min on ice in 1x RIPA buffer (ab156034, Abcam, Cambridge, UK) with a protease inhibitor cocktail (78,430 Thermo Fisher Scientific, Waltham, MA) and stored at − 70 °C. Prior to analysis, the lysate was thawed on ice and centrifuged at 12,000×g, 4 °C for 30 min. The supernatant was transferred to a new tube and stored at − 20 °C as the IC fraction. The conditioned medium was thawed and centrifuged at 100,000×g at 4 °C for 2 h in a fixed angle rotor. The supernatant was collected as the extracellular free-floating protein (FFP) fraction. The pellet was washed once in 500 µl PBS, which had been filtrated twice in a 0.22 µm Millex syringe filter unit (Millipore, Burlington, MA), transferred to a new centrifuge tube, and recentrifuged as above. The ensuing pellet, containing enriched EVs, was resuspended in PBS (0.1% BSA) and split in two equal volumes. These were then diluted in either PBS (0.1% BSA) or in RIPA buffer with the protease inhibitor cocktail (Thermo Fisher Scientific) to generate the two respective EV fractions (RIPA− and RIPA+) (Fig. 1b).
For the uptake experiments, the same protocol as above was used, except that no detergent was applied.

Following treatment, cells were washed on ice with ice-cold 1× PBS with protease inhibitor cocktail (Thermo Scientific). Cell were lysed with lysis buffer containing 1× protease inhibitor cocktail (Thermo Scientific), NaF (4 mM), Na₃VO₄ (20 mM), NaCl (150 mM), β-glycerophosphate (50 mM), and DTT (0.2 mM). Total protein was resolved on 10% SDS-polyacrylamide gels and transferred to PVDF membranes (Fisher Scientific). Membranes were blocked with 5% nonfat dry milk in TBST (50 mM Tris pH 7.6, 150 mM NaCl, 0.1% Tween 20) and incubated overnight at 4 °C in 5% nonfat dry milk in TBST with anti-HIF-1α (1:500, R&D Systems); anti-CAIII (1:500, SCBT), anti-Parp1 (1:1000, Cell Signaling), anti-Cas3 (1:1000, Cell Signaling), or anti-β-tubulin (1:5000, DSHB) antibodies. Specificity of all antibodies has been validated by the manufacturers using siRNA or negative control IgG. Immunolabeling was detected using ECL reagent (LAS4000, GE Life Sciences). Densitometric analysis was performed using ImageQuant TL (GE Life Sciences). All quantitative data is represented as mean ± SE, n ≥ 4 independent experiments.

The immunoblot analysis was performed as described previously.^{15 (link)} Briefly, cells were lysed with RIPA buffer (20–188, Millipore) supplemented with protease inhibitor cocktail and phosphatase inhibitor cocktail (Thermo Fisher Scientific). For the separation of nuclear and cytoplasmic proteins, cells were firstly lysed with cytoplasmic lysis buffer (Tris 10 mM, NaCl 10 mM, MgCl2 3 mM, Nonidet P-40 0.1%) supplemented with protease inhibitor cocktail and phosphatase inhibitor cocktail (Thermo Fisher Scientific) for 3 min, and the supernatant was collected for the detection of cytoplasmic proteins. After three washes with cytoplasmic lysis buffer, the nuclei were lysed with RIPA buffer. Protein concentrations were measured with BCA protein assay kit (Thermo Fisher Scientific). ALKBH5 (HPA007196) and METTL14 (HPA038002) antibodies were from Sigma. YTHDF1 (17479-1-AP) and PTGER4 (66921-1-Ig) antibodies were from Proteintech. METTL3 (ab195352), FTO (ab92821), and Tenascin C (ab108930) antibodies were from Abcam. WNK1 (MA5-35466) and NLRP12 (PA5-89879) antibodies were from Invitrogen. GAPDH (M171-3) and YTHDF2 (RN123PW) antibodies were from MBL. Lamin A/C (4777 S) antibody was from CST. Goat anti-rabbit IgG-HRP (ZB-2301) and goat anti-mouse IgG-HRP (ZB-2305) antibodies were from ZSGB-BIO.