Fig 1: C118P inhibited glutamine metabolism and induced the degradation of ASCT2. MDA-MB-231 and MDA-MB-468 cells were treated with 0.025, 0.05, or 0.1 μM C118P and 10 μM V-9302 for 48 h. Relative ATP production (a), the OCR (b), and relative glutamine uptake (c) were detected, and the data are presented as the means ± S.D. of triplicate measurements and were analysed using Student’s t-test. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. control group. n = 3. (d) ASCT2, GLS1, GLUL, and GDH protein levels in MDA-MB-231 and MDA-MB-468 cells were determined using Western blotting, and β-actin served as a loading control. (e) Cells were treated with 10 μg/mL CHX and vehicle or 0.05 μM C118P for 6/12 h. Then, cell lysates were collected for Western blot analysis of ASCT2. Cells were coincubated with the proteasome inhibitor CQ (20 μM) (f) or MG-132 (1 μM) (g). After pretreatment with C118P (0.05 μM) for 48 h, the expression of ASCT2 was detected by Western blotting. β-Actin served as a loading control. (h) Autophagy-related markers (LAMP-1, LC3 I/II, Beclin1, and p62) were detected after MDA-MB-231 and MDA-MB-468 cells were treated with 0.025, 0.05, or 0.1 μM C118P for 48 h. (i) Autophagosomes in MDA-MB-231 and MDA-MB-468 cells were labelled with MDC. (j) Lysosomes were labelled with LysoTracker Red fluorescence dye. Scale bars, 40 μm. The uncropped bolts are shown in Supplementary Materials.