Fig 2: The SPOP-ASCT2 axis regulates glutamine metabolism, growth and survival of breast cancer cells.a MDA-MB-231 and BT549 cells were transfected with indicated siRNAs and subjected to immunoblotting. b, c MDA-MB-231 cells were transfected with indicated siRNAs for 48 h, followed by assays for glutamine uptake (b) and glutamate production (c) detection (mean ± SD, n = 3). d, e MDA-MB-231 cells were transfected with plasmids expressing wild-type ASCT2 and ASCT2/3 A mutant for 48 h, followed by assays for glutamine uptake (d) and glutamate production (e) detection (mean ± SD, n = 3). f, g MDA-MB-231 cells were transfected with indicated siRNAs and cell viability (f) and clonogenic survival (g) were measured (mean ± SD, n = 3). h MDA-MB-231 cells with stable knockdown of ASCT2, SPOP, along with the shNC control, followed by immunoblotting using indicated antibodies. i-l MDA-MB-231 cells with stable knockdown of ASCT2, SPOP, along with the shNC control were subcutaneously injected to nude mice individually or in combination as indicated. The in vivo tumor growth was measured and plotted (mean ± SEM, n = 7) (i). Tumors were harvested at the end of experiment, photographed (j), weighted (k), and tumor tissues were lysed, followed by immunoblotting (l). m MDA-MB-231 cells were treated with different concentrations of MLN4924 without or with 5 µM V-9302 for 72 h and cell viability was measured by trypan blue exclusion assay (mean ± SD, n = 3). n MDA-MB-231 cells were treated with 5 nM MLN4924, 5 µM V-9302, alone or in combination for 10 days for clonogenic survival (mean ± SD, n = 3). o, p The in vivo growth of MDA-MB-231 xenograft tumors after 14 days of treatment of MLN4924, or V-9302, alone or in combination, along with vehicle control. Tumor growth was monitored (mean ± SEM, n = 10) (o) and the tumor mass were weighted at the end of experiment (p). O-way ANOVA/LSD test for d, e, n, f, g. Two-tailed, unpaired, t test for b, c, k, m, p. Two-way ANOVA/Tukey’s multiple comparison tests for i and o. Source data are provided as a Source Data file.

Fig 3: Glutamine deprivation inhibits SPOP dimerization and promotes SPOP self-ubiquitylation.a, b MDA-MB-231 cellswere cultured with indicated percentage of glutamine for 24 h (a) or glutamine-free medium at different time points (b), for immunoblotting. c MDA-MB-231 cells were cultured in glutamine-free medium for 6 h with MG132 or Chloroquine (CQ) for another 6 h or 24 h, followed by immunoblotting. d MDA-MB-231 cells were transfected with FLAG-SPOP plasmid, cultured in glutamine-free medium with MG132, then for immunoblotting. e, f MDA-MB-231 cells were cultured in glutamine-containing or glutamine-free medium with addition of CHX, harvested at indicated time points for immunoblotting. (e). The band density of SPOP was quantified and normalized to α-tubulin (mean ± SD, n = 3) (f). g HEK293 cells were transfected with indicated plasmids, cultured in glutamine-containing or glutamine-free media for 12 h with or without MG132. Cells were lysed under denaturing conditions, followed by Ni-beads pulldown, and immunoblotting with anti-FLAG antibody. h HEK293 cells were transfected with indicated plasmids, cultured in glutamine-containing or glutamine-free media, followed by immunoprecipitattion with FLAG-agarose beads and analyzed by immunoblotting. i HEK293 cells were transfected with indicated plasmids, cultured in glutamine-containing or glutamine-free media, and lysed under denaturing conditions, followed by Ni-beads pulldown, immunoblotting for ASCT2. j Endogenous SPOP protein in SK-BR-3 cell lysates were pulled down with anti-SPOP antibody, and associated GRK2 were detected by immunoblotting with anti-GRK2 Ab (top, IP). Cell lysates were subjected to immunoblotting (bottom, input). k MDA-MB-231 cells were cultured in glutamine-containing or glutamine-free media in the presence or absence of GRK2 inhibitor PX (10 μM) for 6 h, and analyzed by immunoblotting. l MDA-MB-231 cells were transfected with siRNAs targeting GRK2 or scramble control siRNA, then cultured in glutamine-containing or glutamine-free media for 6 h, and analyzed by immunoblotting. m MDA-MB-231 cells were cultured in glutamine-free medium for indicated time points and analyzed by immunoblotting. n MDA-MB-231 cells were transfected with indicated plasmids, then cultured in glutamine-containing or glutamine-free media for indicated time points, followed by immunoblotting. o–q HEK293 cells were first transfected with siRNA targeting SPOP (3′-UTR) (o, p) then with indicated plasmids (o–q). Cells were cultured with glutamine-free medium for 12 h (o, p) or, cultured in glutamine-containing or glutamine-free media in the absence or presence of PX for 12 h (q), followed by immunoprecipitation with FLAG-agarose beads and analyzed by immunoblotting (o) or lysed under denaturing conditions, followed by Ni-beads pulldown, and analyzed by immunoblotting for FLAG antibody (p) or for ASCT2 (q). Source data are provided as a Source Data file.

Fig 4: 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 5: SPOP interacts with ASCT2.a MDA-MB-231 cells were transfected siRNA targeting Cullin 3, followed by immunoblotting. b Evolutionary conservation of SPOP degron motif on ASCT2. c Schematic diagram of SPOP domains. Shown are breast cancer-associated mutations and cases in MATH domain. d Endogenous SPOP protein in SK-BR-3 cell lysates were pulled down with anti-SPOP antibody, and associated ASCT2 were detected by immunoblotting with anti-ASCT2 Ab (top, IP). Cell lysates were subjected to immunoblotting (bottom, input). e, f HEK293 cells were transfected with indicated plasmids, immunoprecipitated with FLAG-agarose beads (e) or HA antibody (f) and analyzed by immunoblotting. g HEK293 cells were transfected with FLAG-SPOP and indicated HA-ASCT2 plasmids, immunoprecipitated with FLAG-agarose beads and analyzed by immunoblotting. h HEK293 cells were transfected with plasmids expressing either wild type (WT) indicated FLAG-SPOP or E78K mutant, followed by immunoprecipitation with FLAG-agarose beads and analyzed by immunoblotting. Source data are provided as a Source Data file.