FIGURE 13.

Inhibition of TCA-induced ERK1/2 and Akt activation by chemical inhibitors in HuCCT1 cells. HuCCT1 cells were cultured in serum-free medium overnight and pretreated with a selective inhibitor of MEK1/2, U0126 (10 μm), or a selective inhibitor of Akt, MK2206 (3 μm) for 1 h and then treated with S1P (100 nm) or TCA (100 μm) for 1 h. At the end of treatment, total protein was isolated. Protein levels of p-ERK1/2, total ERK1/2, p-Akt, and t-Akt were detected by Western blot analysis. A and C, representative images of the immunoblots for p-ERK1/2, t-ERK1/2, p-Akt, and t-Akt are shown. B and D, relative protein levels of p-ERK1/2/t-ERK1/2 and p-Akt/t-Akt were calculated from three independent experiments. Values represent the mean ± S.E. of three independent experiments. Statistical significance relative to vehicle control: **, p < 0.01, ***, p < 0.001; relative to S1P or TCA treatment group: ##, p < 0.05; ###, p < 0.001.