Fig 2: SPOP shortens ASCT2 protein half-life and promotes ASCT2 ubiquitylation.a MDA-MB-231 cells were transfected with FLAG-SPOP and various mutants for 48 h, followed by immunoblotting. b MDA-MB-231 cells were transfected siRNAs targeting SPOP for 48 h, followed by immunoblotting. c, d MDA-MB-231 cells were transfected with siRNA targeting SPOP (si-SPOP#1) or scramble control siRNA for 48 h, then incubated with cycloheximide (CHX). Cells were harvested at indicated time points for immunoblotting (c). The band density of ASCT2 was quantified using ImageJ software and normalized to a-tubulin (mean ± SD, n = 3) (d). e–h HEK293 cells were transfected with indicated SPOP plasmids (e), indicated ASCT2 plasmids (f), or with indicated ubiquitin mutants (h) lysed under denaturing conditions, followed by Ni-beads pulldown, and immunoblotting for ASCT2 (top panels). The bottom panels are direct immunoblotting using whole cell extracts. Source data are provided as a Source Data file.

Fig 3: CK1d is required for SPOP-mediated ASCT2 degradation.a HEK293 cells were transfected with indicated plasmids, followed by immunoprecipitation with FLAG-agarose beads and immunoblotting. b HEK293 cells were transfected with various FLAG-CK1 isoforms, immunoprecipitated with FLAG-agarose beads and analyzed by immunoblotting. c Endogenous CK1d protein in SUM159 cell lysates were pulled down with anti-S CK1d antibody, and associated ASCT2 were detected by immunoblotting with anti-ASCT2 Ab (top, IP). Cell lysates were subjected to immunoblotting (bottom, input). d MDA-MB-231 cells were transfected with indicated CK1 isoforms, then analyzed by immunoblotting. e MDA-MB-231 cells were transfected siRNA targeting CK1d for 48 h, followed by immunoblotting. f HEK293 cells were transfected with FLAG-SPOP and HA-ASCT2 and treated with D4476 for 12 h before harvesting, followed by immunoprecipitation with FLAG-agarose beads and analyzed by immunoblotting. g MDA-MB-231 cells were treated with different concentrations of CK1 inhibitor D4476 for 24 h, then analyzed by immunoblotting. h, i MDA-MB-231 cells were transfected with si-NC or siRNA targeting CK1d (si-CK1d#1), then incubated with CHX for various time points, and analyzed by immunoblotting ies (h), the band density of ASCT2 was quantified using ImageJ software and normalized to a-tubulin (mean ± SD, n = 3) (i). j HEK293 cells were transfected with indicated plasmids, lysed under denaturing conditions, followed by Ni-beads pulldown, and immunoblotting for ASCT2. Source data are provided as a Source Data file.

Fig 4: Retromer maintains the cell surface abundance and TGN localization of the glutamine transporters ASCT2 and SNAT2, respectively. (A and B) VPS35 knockout HeLa cells were transiently transfected with GFP-VPS35 or GFP alone. After 48 h of incubation, cells were fixed and immunostained with antibodies to endogenous ASCT2 (green) and LAMP1 (red; A) or SNAT2 (green) and LAMP1 (red; B). (C) Colocalization analysis of endogenous SNAT2 (green) and TGN46 (red) in VPS35 knockout HeLa cells transiently transfected with GFP-VPS35 or GFP alone. White asterisks indicate transfected cells. Graphs represent means ± SEM. Pearson’s correlation of three independent experiments with >150 cells per condition; Student’s t test (unpaired). Scale bars, 20 µm and 10 µm in magnified merge panels. ***P < 0.001; ****P < 0.0001.

Fig 5: Inhibitory effects of Ab3-8 on ASCT2 functions. A, Microscopy study of ASCT2 internalization. HEK293 and RH7777 cells overexpressing ASCT2-GFP were reacted with Ab3-8 at 37°C for 1 h. Images were taken at 20 × magnification. Arrows indicate internalized ASCT proteins. Scale bar, 10 µm. B, Quantitative analysis of ASCT2 internalization using FCM. Human CRC cells were incubated with Ab3-8 at 4°C or 37°C for 1 h, followed by PE-conjugated secondary antibody. Numeric data in each panel indicate internalization (%) calculated from the reactivity (MFI) of Ab3-8 with CRC cells at 37°C and 4°C. C, Glutamine-dependent cell growth. Cells were cultured with or without glutamine (2 mmol/L) for 3 d. Relative cell growth was measured every 24 h. Each dot represents the mean of four (HT29) or five (SW1116 and HCT116) independent experiments. D, Intracellular glutamine uptake. Cells were incubated in PBS in the presence of Ab3-8 (30 µg/mL) for 2 h, and then treated with 2 mmol/L glutamine for 10 min. Each dot represents value of individual experiments. E,Cells were incubated in serum- and glutamine-free media in the presence of Ab3-8 for 24 h, and then treated with 2 mmol/L glutamine for 15 min. Total and phosphorylated (p-) AKT and ERK protein levels were measured by western blotting. GAPDH was used as a loading control. Each dot represents value of individual experiments. Data are expressed as mean ± SD of at least four independent experiments