FIGURE 5.

Effects of Ag120 on glutamine metabolism and proliferation in CRC cells following ASCT2/SLC1A5 knockdown or overexpression. (A,B) Immunoblot analysis of ASCT2 in HCT116 and HT29 cells stably transfected with ASCT2 shRNA (shSLC1A5#1/shSLC1A5#2) or control shRNA (A) or stably transfected with a control or SLC1A5 DNA-lipid overexpression complex (B). (C,D) Glutamine uptake in SLC1A5-depleted or SLC1A5-overexpressing HCT116 and HT29 cells treated for 2 h with Ag120 (15 µM). The contribution of Ag120 to glutamine uptake was calculated based on the data in Supplementary Figures S4C,D. (E,F) Viability of SLC1A5-depleted or SLC1A5-overexpressing HCT116 and HT29 cells treated for 72 h with Ag120 (45 µM) was quantified using a CCK-8 kit. The contribution of Ag120 to cell viability was calculated based on the data in Supplementary Figures 4E,F. (G,H) As described for (E,F) except colony formation was measured after 10 days. Mean ± SD of three experiments. *p < 0.05, **p < 0.01 by Student’s t-test.