Fig. 2

Effect of PKC activation on alanine uptake activity in Caco-2 cells. a hLAT1 and hLAT2 mRNA expression levels in Caco-2 cells were examined by qPCR. The values were normalized to the hGAPDH mRNA expression level, and the data are shown as the mean ± SD of values obtained from three separate experiments, each performed in duplicate. b Functional expression of hLATs in Caco-2 cells was examined by a transport assay. The cells were incubated with alanine (50 µM) in Na⁺-free HBSS buffer for 5 min at 37℃. Overloaded alanine (10 mM) and BCH (1 mM) were used as inhibitors for alanine uptake-related transporters and LATs, respectively. Its solvent (0.1% (v/v) sterilized water) was used as a control (-). Data are shown as the mean ± SD of values obtained from three separate experiments, each performed in duplicate. **, p < 0.01 vs control (-); NS, not significant. c, d The effect of PKC activation on alanine uptake activity in Caco-2 cells was examined by a transport assay. To activate PKC, the cells were treated in the absence (0.1% (v/v) DMSO, -) or presence of PMA (0.1 or 1 µM) for 4 h. To determine whether the PMA-induced effect depends on PKC activation, the cells were exposed to Go6983 (1 µM) or its solvent (0.1% (v/v) DMSO, -) at the same timing as the PMA treatment. After removing the reagents, alanine uptake was conducted in the same way as explained in b. d To elucidate the alanine uptake activity by hLAT1, an LAT1-specific inhibitor, JPH203 (10 µM), was added to the substrate solution. Data are shown as the mean ± SD of values obtained from three separate experiments, each performed in duplicate. *, p < 0.05; **, p < 0.01 vs -/- (PMA/Go6983), ##, p < 0.01 vs 0.1 µM/-, †, p < 0.05; ††, p < 0.01 vs 1 µM/-