Figure 1.

Knockout of ASCT2 strongly decreases glutamine transport rate but does not alter LAT1 expression and activity. A, the LAT1 and ASCT2 protein expression levels were analyzed by immunoblotting in LS174T and A549 WT, ASCT2^KO and LAT1^KO cells demonstrating an independence of protein expression between ASCT2 and LAT1. Two independent clonal cell lines of ASCT2^KO are shown (#1 and #2). Tubulin was used as a loading control. B, glutamine transport rates for WT and ASCT2^KO#1 cells were measured by [¹⁴C]-glutamine uptake in HBSS media containing 10 μm glutamine. Inhibitors of system A (MeAIB) (10 mm) or LAT1 (JPH203) (30 μm) were used to identify transporters responsible for the residual glutamine transport activity in ASCT2 KO cells. C, LAT1 transport activity of WT and ASCT2^KO#1 cells was measured by [¹⁴C]-leucine uptake in Na⁺-free HBSS media containing 10 μm leucine with (white) or without (dark gray) the LAT1 inhibitor JPH203 (30 μm). All of the above results represent the average of at least three independent experiments. *, significant compared with WT (p < 0.05); #, significant compared with untreated ASCT2^KO (ANOVA, p < 0.05); n.s., not significant.