Tsg101

Total proteins were extracted by RIPA lysis buffer with proteinase inhibitor (Roche, Switzerland). An equal amount of total protein (20–40 μg) was separated by SDS-PAGE (Beyotime Biotechnology, Shanghai, China), transferred into the PVDF membrane (Millipore, USA), and then incubated overnight with primary antibodies specific for HIF1AN (Abcame, USA), β-tubulin (Proteintech, China), CD9 (Abcame, USA), CD81 (Abcame, USA), HSP70 (Abcame, USA), TSG101 (ABclonal, China). Then, the membrane was incubated with secondary antibodies (Aspen, China) for 1 h and exposed to X-ray film (UVP, USA).

The human colorectal cancer cell line, HCT8, was purchased from the National Collection of Authenticated Cell Cultures (Shanghai, China) and cultured in RPMI-1640 (Gibco, MA, USA) containing 10% FBS (Gibco). The 5FU resistant cell line, HCT8FU, was purchased from OBIO Technology (Shanghai, China) and maintained in a medium containing 0.5 μg/ml 5FU. The IDH1 gene was cloned into a pLenti-CMV-EGFP-3FLAG-Puro vector and stably expressed in HCT8 cells using a lentivirus. The lentivirus of shRNA-NC (sense 5'-GATCCGTTCTCCGAACGTGTCACGTTTCAAGAGAACGTGACACGTTCGGAGAACTTTTTTG-3', antisense 5'-AATTCAAAAAAGTTCTCCGAACGTGTCACGTTCTCTTGAAACGTGACACGTTCGGAGAACG-3'), shRNA-IDH1-994 (sense 5'-GATCCGCAGTACAAGTCCCAGTTTGATTCAAGAGATCAAACTGGGACTTGTACTGCTTTTTTG-3, antisense 5'-AATTCAAAAAAGCAGTACAAGTCCCAGTTTGATCTCTTGAATCAAACTGGGACTTGTACTGCG-3') and shRNA-IDH1-1404 (sense 5'-GATCCGCTTCATGACCAAGGACTTGGTTCAAGAGACCAAGTCCTTGGTCATGAAGCTTTTTTG-3', antisense 5'-AATTCAAAAAAGCTTCATGACCAAGGACTTGGTCTCTTGAACCAAGTCCTTGGTCATGAAGCG-3') were used for stable IDH1 knockdown. 5FU and Ivosidenib were purchased from Selleck Chemicals (Shanghai, China). The antibodies, anti-Flag (Sigma, MO, USA), IDH1 (R&D Systems, MN, USA), CD63 (Proteintech, Wuhan, China), TSG101 (ABclonal, Wuhan, China), and β-actin (Cell Signaling Technology, MA, US) were also used in this study.

After the total protein loading of each EV sample was made consistent by silver staining, the total proteins were then separated by SDS-PAGE; transferred to PVDF membranes; determined with antibodies, including CD63 (Cat#A19023, ABclonal, Wuhan, China), TSG101 (Cat#A5789, ABclonal, Wuhan, China), HSP70 (Cat#A0284, ABclonal, Wuhan, China), calnexin (Cat#A4846, ABclonal, Wuhan, China), ApoA1 (Cat#A4163, ABclonal, Wuhan, China), ApoA4 (Cat#5700S, Cell Signaling Technology, Danvers, MA, USA), ApoE (Cat#A0304, ABclonal, Wuhan, China), ApoB100 (Cat#A4184, ABclonal, Wuhan, China), albumin (Cat#A11625, ABclonal, Wuhan, China), human IgG (Cat#A19711, ABclonal, Wuhan, China), and human IgM (Cat#A19719, ABclonal, Wuhan, China); and detected using the ECL system (LABLEAD, Beijing, China).

Cell culture medium and human primary renal PTECs were provided by ScienCell (San Diego, CA, United States). Anti-SLC2A9 (URAT1) antibodies, TSG101, CD63, caspase-1, GSDMD, IL-1β, caspase-3, caspase-8, caspase-9, cytochrome c, Bcl-2, and Bax were purchased from ABclonal (Wuhan, China). Oxonic acid (OA) and UA were provided by Sigma (St. Louis, MO, United States). CIAS1/NALP3, GAPDH, and anti-GLUT9 were provided by Abcam (Cambridge, United Kingdom). In addition, the CCK-8 assay kit was provided by Jiwei Biological Technology (Shanghai, China).

Protein was extracted from an exosome using a Total Exosome Protein Isolation Kit (ThermoFisher). The cultured cells were lysed in Radio Immunoprecipitation Assay Lysis Buffer (Biosharp). The protein sample was separated using sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE, Bio‐Rad) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore). The following antibodies were used as primary antibodies: Alix (ab186429, Abcam), TSG101 (A1692; Abclonal), CD63 (A19023, Abclonal), HSP70 (A12948; Abclonal), phospho‐PI3K (AP0854, Abclonal), PI3K (A11526, Abclonal), phospho‐Akt (AP0637, Abclonal), Akt (A11016, Abclonal), phospho‐FoxO1 (AP0172, Abclonal), FoxO1 (A2934, Abclonal), phospho‐FoxO3a (AP0684, Abclonal), FoxO3a (A0102, Abclonal), and β‐actin (AC026, Abclonal). Secondary antibodies were horseradish peroxidase (HRP) goat anti‐rabbit IgG (AS014, Abclonal) and goat anti‐mouse antibodies IgG (AS003, Abclonal). Visualization and analysis were performed using the iBright1500 system (Thermo).

The frozen renal cortex samples were ground by a Lu Ka sample freezing grinder (LUKYM-I, Guangzhou). Equal amounts of protein were separated by SDS-PAGE and transferred to PVDF membranes. Then, the membranes were incubated with 10% BSA in Tris/Tween-buffered saline and primary antibodies against LDHA (19987-1-AP; Proteintech, USA), HK2 (22029-1-AP; Proteintech, USA), HIF-1α (20960-1-AP; Proteintech, USA), CD63 (A5271; ABclonal, USA), TSG101 (A2216; ABclonal, USA), and β-actin (FD0060; Fdbio Science, China) overnight. Bands were detected with an ECL Plus western blotting detection system (GelView 6000 Pro, Guangzhou, China) and analyzed with Quantity One software (Bio-Rad, CA, USA).

EVs or tissue samples were lysed with radioimmunoprecipitation assay (RIPA) and proteinase inhibitor cocktail (Servicebio, Wuhan, China) at 4°C for 30 minutes. Then the lysates were centrifuged at 12,000 g for 20 minutes at 4°C. The protein concentration of EVs and Fallopian tube tissue samples were measured using a BCA Protein Assay Kit (Servicebio, Wuhan, China). 5μg of each sample was loaded for electrophoresis, using SDS/PAGE (10% gel), followed by transferring to PVDF membrane (Merck Millipore, Burlington, MA, USA). Then, the membrane was blocked using 5% skimmed milk for 1 h at room temperature, followed by washing with TBS for 15 min. Afterward, the membrane was incubated with the primary antibodies at 4 °C overnight. The primary antibodies used for immunostaining were TSG101 (1:1000; Abclonal, Woburn, MA, USA) and CD9 (1:1000; Abcam, Cambridge, UK), and the secondary antibodies were goat anti-rabbit labeled by horseradish peroxidase (1:2000; Servicebio, Wuhan, China). The membrane was incubated with the secondary antibodies for 1h at 37 °C and then was immersed in an electrochemiluminescence (ECL; Absin, Shanghai, China). The signals were detected using a Gene Gnome XRQ chemiluminescence imaging system (Syngene, Bengaluru, India).