Sml1767

For the stimulation experiments, IVD cells were cultured for 24 h in a 6-well plate at a density of 2 × 10⁵ cells/well in DMEM/F12 supplemented with 1% Antibiotic-Antimycotic and 10% FCS. On the next day, cells were serum starved in media supplemented with Polymyxin B (100 units/mL, 81271, Sigma-Aldrich) with or without Sparstolonin B (SsnB, 25 μM, TRL-2/TRL-4 inhibitor, SML1767, Sigma-Aldrich) for 2 h prior to stimulation with FnF. Afterwards, cells were treated with 30 kDa proteolytic fragments from human plasma fibronectin (FnF, 500 nmol/L, F9911, Sigma-Aldrich) or recombinant canine IL-1β, which was used as positive inflammation control (10 ng/ml, 3747-CL-025, R&D Systems, Inc., Minneapolis, MN 55413, USA) for 18h. Untreated NP cells served as a negative control group, NP cells treated with Sparstolonin B alone were used as a control to the combination treatment of FnF, and Sparstolonin B. Used concentrations were based on the range of FnF found in IVDs during degeneration (19 (link), 29 (link)). After the incubation time, media supernatants were collected, and cells were lysed using RLT Lysis Buffer (79,216, Qiagen, Hilden, Germany). Both cell lysates and supernatants were stored at −80°C until further analysis.

To assess the influence of co-culturing IVD cells and C. acnes and of treatment with sparstolonin B (SML1767, Sigma, St. Louis, MO, USA) on metabolic activity, the MTT assay (3-[4–dimethylthiazol-2-yl]μ-2,5-diphenyl tetrazolium bromide formazan, M2003, Merck, Darmstadt, Germany) was used. IVD cells were seeded and co-cultured with C. acnes (MOI 1, 10, and 100) with or without stimulation of different sparstolonin B concentrations (10, 15, 20, 25, 50, and 100 μM). Extracellular bacteria were killed by incubation for 2 h with penicillin G (0.128 mg/mL, 13752, Merck, Darmstadt, Germany). The cells were then incubated with MTT in DMEM/F12 (5 mg/mL) for 2 h at 37 °C, 0.04 M HCl (H1758, Sigma, St. Louis, MO, USA) in isopropanol (2-propanol, I9516, Sigma, St. Louis, MO, USA) was used as a solubilization solution, and the absorbance was measured at 570 nm. Metabolic activity was calculated relative to the untreated control.