I2273

Serum samples (100 µL) and prechilled methanol (400 µL) were mixed by well vortexed. Cecal contents, liver, and adipose tissues (≈10 mg) were individually grounded with liquid nitrogen and the homogenate was resuspended with prechilled 80% methanol and 0.1% formic acid by well vortexed. The samples were incubated on ice for 5 min and then were centrifuged at 15 000 rpm, 4 °C for 5 min. The supernatants were diluted to final concentration containing 53% methanol by LC‐MS/MS grade water. The samples were subsequently transferred to a fresh Eppendorf tube and then were centrifuged at 15 000 g, 4 °C for 10 min. The supernatant was analyzed for tryptophan, I3AA, and other indole derivatives using 4000 Q TRAP LC‐MS/MS (AB Sciex) analysis according to a previously described method (Table S5, Supporting Information).^[33 (link)
^] Specifically, L‐tryptophan (T0254, Sigma), I3AA (45533, Sigma), indole (442619, Sigma), 3‐indoleacrylic acid (I2273, Sigma), indole‐3‐carboxaldehyde (129445, Sigma), and indole‐3‐lactic acid (I157602, Aladdin) were used as standards in the current study. The absolute concentration of metabolites was calculated according to the standard curves, which were created using six appropriate serial dilutions of the corresponding standards.

The cell cultivation was completed in a 6 mm plate; the number of cells accounted for roughly 30–40% of an orifice. We used 2 μΜ of drug and 0.1% DMSO. Each concentration had triplicate experiments over 48 h. Cells were resuspended in lysis buffer combined with a 1 X PMSF proteinase inhibitor cocktail (Thermo Scientific, USA). The total protein concentration was measured using Thermo Scientific NanoDrop One (protein A280). Then, equal amounts of proteins (approximately 1 mg) were diluted with 450 µL of NH₄HCO₃ (50 mM) and added to Ultra filtration tubes (Amicon Ultra-4, PLGC Ultracel-PL ultrafiltration membrane, 10 kDa), and we added a 10 mM DTT reduction (A2948; AppliChem), 20 mM IAA alkylation (I2273; Sigma). Each sample was centrifuged at 14,000× g to pass the solution through a filter. The resuspension was added before the total intracellular protein was hydrolyzed by trypsin (90057; Thermo Scientific™) at 37 °C (12–14 h) and desalted using a Thermo Scientific Pierce C18 tip. We freeze-dried samples and used 1% TFA resuspensions (see Supplementary Materials for details). They were analyzed by mass spectrometry (Orbitrap Fusion Lumos, Shanghai, China). Screening for significantly different proteins was analyzed by DAVID (https://david.ncifcrf.gov (accessed on 12 March 2021)), STRNG (https://string-db.org (accessed on 12 March 2021)), and Cystoscope [50 (link)].