Parthenolide

Parthenolide (pan-inflammation inhibitor: NFκB, AP-1, and NALP3 inflammasome pathway inhibitor) and ACHP (NFκB specific inhibitor) were purchased from TOCRIS (0610, 4547, Bristol, UK). Indomethacin (COX1/2 inhibitor) was purchased from Sigma-Aldrich (I7378, St. Louis, MO, USA). CLI-095 (TLR4 inhibitor) was purchased from InvivoGen (tlrl-cli95, San Diego, CA, USA). Fluoranthene was obtained from AccuStandard (H-118N, purity 97.2%, New Haven, CT, USA) and 1-methylanthracene from Crescent Chemical (DRE-C20834900, purity 99.5%, Islandia, NY, USA). Glyburide (Glybenclamide; NLRP3 inhibitor) was purchased from Novus (NBP2-30141, Centennial, CO, USA). All inhibitors and PAH were dissolved in DMSO (Thermo Fischer Scientific, Waltham, MA, USA), as done previously [36 (link),37 (link),40 (link)]. Recombinant mouse TNF-alpha (rTNF) was purchased from R&D systems (410-MT, Minneapolis, MN, USA).

The kinase inhibitor of NF-κB-1 (KINK-1; 5 μM), IKKβ inhibitor, was procured from MedChemExpress (Monmouth Junction, NJ, USA). The cycloheximide (CHX; 5 µM) was purchased from Sigma-Aldrich (St. Louis, MO, USA) and MG132 (10 Μm) was procured from Selleck Chemicals (Houston, TX, USA). The parthenolide (5 μmol/L), p65 inhibitor was purchased from Tocris Bioscience (Ellisville, Missouri, USA). LPS used for this study was obtained from Sigma-Aldrich (Saint-Louis, Missouri, USA).

NF-κB inhibitory activity was assessed using a HEK293/NF-κB-luc cell line as previously described [26 (link)]. Briefly, cells were incubated 1 h in FBS-free medium with 2.5 µM of Cell Tracker Green CMFDA (Thermo Fisher Scientific), a fluorescent dye used to measure cell viability, and seeded in 96-well plates (10⁴ cells/well). After an overnight incubation, cells were treated with patulin or vehicle only (0.5% DMSO in culture medium) and stimulated with TNF-α (20 ng/mL) (Sigma-Aldrich) for 5 h. Then, the cells were lysed with reporter lysis buffer (Promega, Madison, WI, USA) and both fluorescence of the Cell Tracker Green CMFDA and luminescence of the firefly luciferase were read on a Cytation 3 imaging multimode reader (Biotek, Winooski, VT, USA). The luminescence signal was normalized by the fluorescence signal for each well, and relative NF-κB activity was quantified by comparing the normalized luminescence signal of sample-treated cells with the vehicle-treated cells. Nonlinear regression (with sigmoidal dose response) was used to calculate the IC₅₀ values using GraphPad Prism 6.05. Each compound was tested in duplicate and three independent experiments were performed. Parthenolide (Tocris Bioscience, Bristol, UK) was used as a positive control. Extracts and fractions were screened at 20 μg/mL. Patulin dose–response curve started at 10 μM.