Figure 2.

Interleukin-6 (IL-6) and methotrexate (MTX) reduced SLC19A1 expression and uptake of MTX into mouse synoviocytes. Synoviocytes from arthritic mice were cultured for 24 h with (A) IL-6 (1, 10, 100 ng/mL) + soluble IL-6 receptor (sIL-6R) (100 ng/mL), (B) MTX (1, 10, 100 nM), (C) tumor necrosis factor -α (TNF-α) (0.1, 1, 10 ng/mL), or (D) IL-6 (100 ng/mL) + sIL-6R (100 ng/mL), MTX (100 nM), or IL-6 (100 ng/mL) + sIL-6R (100 ng/mL) + MTX (100 nM). After culturing, cell lysate was collected and the mRNA expression for SLC19A1 was measured by real-time polymerase chain reaction. (E) Mouse synoviocytes were cultured for 24 h with IL-6 (100 ng/mL) + sIL-6R (100 ng/mL), MTX (100 nM), or IL-6 (100 ng/mL) + sIL-6R (100 ng/mL) + MTX (100 nM). The uptake of Alexa Fluor 488 conjugated MTX (100 nM) was quantified by measuring the fluorescence emission for each sample. Results are expressed in arbitrary units of fluorescence intensity. Each column and vertical line represents the mean and standard deviation of triplicate cultures. Statistical significance was analyzed by Dunnett's multiple comparison test (#P < 0.05) or unpaired t-test (* P < 0.05).