Indole

HCT-8 cells were pre-treated with indole (Acros Organics, New Jersey, NJ) at 3 concentrations (0.25, 0.5 and 1mM) for 4 hours prior to TNF-α stimulation. The maximum indole concentration as based on our prior work (Kohli et al., 2018 (link)) as well as observations from a human study showing an average indole concentration of ~2.5 mM in the fecal stream (Hoffman et al., 2015 (link)). A 50 mM stock solution of indole was initially prepared in N,N-dimethylformamide (DMF) and subsequently diluted in growth medium to achieve the desired final concentrations. Control cells (no indole) were exposed to the same volume of DMF as the corresponding indole concentration. After 4 hours, TNF-α was added to the wells and the cells were exposed to TNF-α in the presence of different concentrations of indole. Three concentrations (0.1, 1, and 10 ng/mL) of recombinant human TNF-α (R&D Systems, Minneapolis, MN, USA) diluted in growth medium were used. The maximum concentration of TNF-α was based on the ability to elicit a strong inflammatory response without inducing apoptosis (Krishnan et al., 2018 (link)). All experiments were performed in triplicate.

DAcPC was synthesized and purified as described previously.^{49 (link)} All solvents were procured from commercial vendors through VWR International, Inc. and used as received: chlorobenzene and n-hexadecane (99%) from Alpha Aesar, ethanol (ACS grade) from BDH, and dry solvents acetonitrile, benzene, chloroform (ethanol stabilized), dichloromethane, 1,2-dichloroethane, 2-propanol, N,N-dimethylformamide, benzene, ethyl acetate, methanol, tetrahydrofuran, and dimethylsulfoxide (DMSO) from EMD Millipore. Used water was prepared in-house using a Barnstead Nanopure™ TOC Life Science ultrapure water system. The used fluorescent dyes, indole and 9-diethylamino-5-benzo[α]phenoxazinone (Nile Red, NR), were obtained from Acros.

Unless specified, chemicals used for protein expression and purification were purchased from Fisher, Sigma, RPI, or GoldBio and were used without further purification. Reagents used for crystallization were purchased from Hampton Research. l-Tryptophan (99%), d-tryptophan (99%), indole (99+%), indoline (98%), benzothiazole (97%), benzoxazole (99+%), benzimidazole (98%), aniline (99+%), l-glutamine (99%), and l-asparagine (99%) were purchased from Acros Organics. Dimethylallyl diphosphate (DMAPP) (>95%) and dimethylallyl S-thiolodiphosphate (DMSPP) (>95%) were purchased from Echelon Biosciences.