Celecoxib

Lifespan assays were performed on nematode growth media (NGM) plates. Synchronized L4 or young adult worms were transferred to 5 NGM plates (6 cm diameter). A total of 20–30 worms were transferred on to each NGM plate containing 40 µM FUDR. All lifespan assays were performed at 20°C. The worms were scored as live, dead, or lost and transferred to new NGM plates every other day. Worms that failed to display touch-provoked movement were scored as dead. For celecoxib treatment, 500 µL of 10 µM celecoxib (Sigma-Aldrich, St. Louis, MO, USA) was added on to the surface of NGM media 8 hours before transferring worms onto the plates. celecoxib was dissolved in DMSO for storage and diluted in water to work concentration before use. After adding the compound to the NGM plates, the final DMSO concentration was 0.1%. Control plates contained 0.1% DMSO too. All lifespan tests were repeated three times. Survival curves and statistical analyses were carried out using SPSS software (SPSS, Chicago, IL, USA). P values were calculated using the log-rank test.

To administer a neutralizing antibody specific to TNF-α (Anti-TNF), mice receiving either LF41 or PBS challenge were IP-injected every one day with Anti-TNF-α (0.2 mg/kg; R&D Systems) or its isotype IgG1 (0.2 mg/kg; R&D Systems), from day 1 to day 9.
To perform in vivo inhibition of activity of COX-2 or EP-4, mice orally receiving LF41 or PBS were given daily IP injection with a COX-2-specific inhibitor celecoxib (6 mg/kg; Sigma) or daily IG inoculation of a EP-4-specific inhibitor ONA-AE3-208 (I-EP4) (5 mg/kg; ApexBio, Boston, MA), from day 1 to day 10.
For administration of an IL-10-specific neutralizing antibody (Anti-IL-10) to evaluate its effect on LPS-induced serum ALT levels, mice pretreated with PBS or H-LF41 for 10 days were given IP injection with Anti-IL-10 (0.25 mg per mouse; BD Bioscience Pharmingen) or its isotype control IgG1 (0.25 mg per mouse; BD Bioscience Pharmingen) 30 minutes prior to LPS challenge.
In some experiments where mice were not inoculated with LPS, these mice were given IP inoculation of Anti-IL-10 (0.4 mg/kg) or its isotype control IgG1 (0.4 mg/kg) every one day, from day 1 to day 9.
ONO-AE3-208 (I-EP4) was dissolved in 0.003N NaOH, and celecoxib in 0.5% methylcellulose (Sigma). The dosage and vehicles for ONO-AE3-208 and celecoxib were guided by earlier reports [23 (link)–24 (link)].

IL-1β, purchased from GenScript (Z02922-10; Picataway, NJ, USA), was dissolved in sterilized water. Obtusifolin and celecoxib were purchased from Sigma-Aldrich (Obtusifolin, PHL83880; celecoxib, PZ0008; St. Louis, MO, USA). Primary mouse chondrocytes were incubated at 37 °C with 5% CO₂ for 4 days. Thereafter, the cells treated with IL-1β (1 ng/mL) were co-treated with Obtusifolin for 24 h and harvested on day 5. The cells were also co-treated with celecoxib and IL-1β (1 ng/mL). To identify signaling pathways regulated by Obtusifolin, chondrocytes were pretreated with Obtusifolin for 24 h. IL-1β (1 ng/mL) was added to the cells, and they were incubated for 10 min in serum-free medium before harvest.

To prepare 1-MT for oral gavage, 1 g of 1-dl-MT (MilliporeSigma) was added to a 15 ml conical tube with 7.8 ml Methocel/Tween [0.5% Tween/0.5% Methylcellulose (v/v in water) (MilliporeSigma)]. The following day, the 1-MT concentration was adjusted to 85 mg/ml by adding an additional 4 ml Methocel/Tween and mixing again briefly. For in vitro use, 1-MT was prepared as a 20 mM/L stock in 0.1 N NaOH, adjusted to pH 7.4 and stored at -20°C protected from light. To prepare indomethacin (MilliporeSigma), a stock solution was made at a concentration of 50 mg/ml in 100% ethanol, and heated to dissolve. Thereafter, indomethacin stock solution was diluted 1:10 in 25% Solutol. Celecoxib (MilliporeSigma) was prepared by dissolving 100 mg in 0.5 ml of DMSO for 2–3 hours at 37°C, creating a stock solution of 200 μg/μL. Stock Celecoxib was diluted 1:100 in water. AH6809 (Cayman Chemical) was prepared by dissolving 1 mg into 500 μL of Solutol (heated at 60°C in order to get into solution).