FIGURE 3.

A and B, effect of TCA on COX-1 expression. HuCCT1 cells were cultured in serum-free medium overnight and then treated with S1P (100 nm) or TCA (100 μm) with or without JTE-013 (10 μm) for 8 h. At the end of treatment, total proteins were isolated. The protein level of COX-1 was detected by Western blot analysis. A, representative images of immunoblots for COX-1 and Actin are shown. B, relative protein levels of COX-1 were analyzed using Actin as a loading control. Values represent the mean ± S.E. of three independent experiments. C and D, effect of TCA on inflammatory cytokines expression. HuCCT1 cells were cultured in serum-free medium overnight and then treated with S1P (100 nm) or TCA (100 μm) with or without JTE-013 (10 μm) for 8 h. At the end of treatment, total cellular RNA was isolated. Relative mRNA levels of IL-6 (A) and TNF-α (B) were determined by real-time RT-PCR and normalized using GAPDH as an internal control. Values represent the mean ± S.E. of three independent experiments. Statistical significance relative to the vehicle control; *, p < 0.05; **, p < 0.01; ***, p < 0.001; relative to S1P or TCA treatment group: #, p < 0.05; ##, p < 0.01; ###, p < 0.001.