Fig. 4.

Substrate determination and cytotoxicity characterization of predicted ligands. Predicted LAT-1 ligands validated in cis-inhibition assays were subjected to substrate determination by trans-stimulation of L-leucine efflux (1 μM unlabeled and 10 nM radiolabeled). (A) Cells were preloaded with L-leucine, and efflux was induced by subsequent addition of each test compound at a concentration of 1 mM. Gabapentin, L-leucine, and BCH were included as positive controls, and glycine and guanfacine were included as negative controls. (B) The cytotoxic effects of acivicin (100 μM) and 3-iodo-L-tyrosine (1 mM) against T98G glioblastoma cells stably expressing an shRNA against LAT-1 (T98G-KD; filled bars) or EV (T98G-EV; open bars) are depicted. Cell proliferation for both cell lines and treatment conditions were normalized to cell density at treatment day 0 and then to the vehicle control treatment at 48 h. Each bar represents the mean of three or four separate experiments, and error bars represent the SEM. Statistical analysis in A was by one-way ANOVA and Dunnett’s multiple comparison test and in B was by two-way ANOVA and Bonferroni correction for multiple testing. *P < 0.05.