Effects of methotrexate on the prostaglandin production by rheumatoid arthritis fibroblast-like synoviocytes (FLSs). Synovial fibroblasts were left untreated or activated with 10 ng/mL interleukin-1-beta (IL-1β) in the absence or presence of 10 or 250 μM methotrexate for 48 hours. After treatment, total protein was isolated and assayed for enzyme expression, while β-actin was used as loading control. Supernatants were analyzed for prostanoid content by using liquid chromatography tandem mass spectrometry. (a) Analysis of prostaglandin E2 (PGE2) formation in control and IL-1β-stimulated FLSs with or without methotrexate. (b) Representative Western blot results show expression of microsomal prostaglandin E2 synthase 1 (mPGES-1), cyclooxygenase 2 (COX-2), and 15-prostaglandin dehydrogenase (15-PGDH) in rheumatoid arthritis FLSs. (c) Analysis of PGD2, PGF2α, and the metabolites of PGI2 and TxA2 (6-keto PGF1α and TxB2, respectively) in culture supernatants indicates the amount formed after 48 hours of incubation. Horizontal lines indicate median values, and whiskers indicate range values (n = 4). Ctrl, control; Mtx, methotrexate; Tx, tromboxane.