(A, B) V5-tagged SLC1A5 (SLC1A5-V5) (A) or V5-tagged SLC38A2 (SLC38A2-V5) (B) was transiently co-expressed with Flag-tagged RNF5 WT, RNF5 RING mutant (RM), or RNF5 lacking its C-terminus (ΔCT) in H293T cells. Cells were treated (10 μM MG132, 4 hr) and cell lysates were subjected to immunoprecipitation (IP) followed by western blotting (WB) with indicated antibodies. (C, D) SLC1A5-V5 (C) or SLC38A2-V5 (D) was co-expressed in H293T cells with HA-Ub plus indicated RNF5 constructs. Cells were treated with MG132 (10 μM, 4 hr) prior to preparation of lysates, subjected to IP followed by WB using indicated antibodies. Asterisk = IgG heavy chain. (E, F) H293T cells expressing SLC1A5-V5 (E) or SLC38A2-V5 (F) were transfected with increasing amounts of Flag-RNF5 WT and treated as indicated with MG132 (10 μM, 4 hr). WB performed with indicated antibodies. (G, H) H293T cells were transfected with SLC1A5-V5 and either a 3XFlag-tagged control construct, or indicated RNF5 constructs and treated with cycloheximide (CHX; 50 μg/ml) for indicated times. WB performed with indicated antibodies (G) and SLC1A5 protein levels were quantified by densitometry (H). Bars represent mean values ± s.d. of three experiments. See also .

(A) MDA-MB-231 cells were treated with 2.5 μg/ml BFA, 5 μg/ml tunicamycin, or 2 μM thapsigargin for indicated times. WB performed with indicated antibodies. (B) MDA-MB-231 cells were treated with BFA (2.5 μM, 24 hr) in the absence or presence of MG132 (10 μM, 4 hr). WB performed with indicated antibodies. (C) MDA-MB-231 cells were incubated with BFA (2.5 μg/ml, 12 hr) and then treated with cycloheximide (CHX; 50 μg/ml) for indicated times. Left panel: WB performed with indicated antibodies. Right panel: SLC1A5 protein levels were quantified by densitometry. Bars represent mean values ± s.d. of three experiments. (D-F) Cell lysates were prepared from MDA-MB-231 cells treated with 2.5 μg/ml BFA (D) or 100 nM paclitaxel (E) or transfected with indicated shRNA and incubated with 2.5 μg/ml BFA (F) for indicated times and treated with MG132 for 4 hr before preparation of cell lysates. IP and WB were then performed with indicated antibodies.. * = IgG heavy chain. (G) MDA-MB-231 cells were transfected with shControl or shRNF5 and incubated with 2.5 μg/ml BFA, 5 μg/ml tunicamycin, or 2 μM thapsigargin for indicated times. Left panel: WB performed with indicated antibodies. Right panel: SLC1A5 protein levels were quantified by densitometry. Bars represent mean values ± s.d. of three independent experiments. See also .

(A) MDA-MB-231 cells transfected with indicated constructs were incubated for 6 hr with media lacking Gln, followed by media supplemented with 1 mM Gln for 15 min before cells were harvested. Levels of intracellular L-Gln were quantified using GC-MS. Bars represent the mean ± s.d. (B) MDA-MB-231 cells were incubated with 100 nM paclitaxel for 24 hr and then subjected to incubation in medium containing 1 mM ¹³C-glutamine (50% labeled). Intracellular ¹³C-glutamine and ¹³C-glutamine-derived metabolites were measured by GC-MS. Bars represent the mean ± s.d. (C) MDA-MB-231 cells transfected with indicated constructs were treated with paclitaxel (100 nM for 18 hr), and BCH (10 mM for 1 hr prior to addition on radiolabeled Gln), and then incubated in medium containing ³H-Gln (5 μCi/ml; 15 min). Duplicate samples were subjected to liquid scintillation assay. Bars represent the mean ± s.d. (D) MDA-MB-231 cells transfected with indicated constructs were incubated for indicated times with or without 2.5 μg/ml BFA. WB performed with indicated antibodies (E) MDA-MB-231 cells transfected with the indicated constructs were incubated with or without BFA for 24 hr. Viable cells were counted using trypan blue exclusion. Bars represent the mean ± s.d. of three independent experiments. (F). MDA-MB-231 cells transfected as in panel E were incubated with or without BFA for 7 day. Colonies were stained with crystal violet and counted. Bars represent the mean ± s.d. of three independent experiments. (G) MDA-MB-231 cells transfected with shControl or shRNF5 were treated with BFA in the absence or presence of rapamycin (1 μM for 12 hr). WB performed with indicated antibodies. (H) MDA-MB-231 cells transfected and incubated with BFA in the absence or presence of rapamycin as in panel G. One week later, colonies were stained with crystal violet and counted. Bars represent means ± s.d. of three independent experiments. See also .

(A) Cell lysates from BCa lines MCF7, MDA-MB-231 (231), MDA-MB-453 (453), MDA-MB-468 (468), SKBR-3, or T47D were immunoblotted with indicated antibodies. (B) Indicated cell lines were incubated with 20 μM cisplatin (Cis), 0.5 μg/ml doxorubicin (Dox) or 100 nM paclitaxel (Pacl) for 24 hr. WB performed with indicated antibodies. (C) Indicated cell lines were incubated with doxorubicin or paclitaxel for indicated times. WB performed with indicated antibodies. (D) Indicated cell lines were incubated with paclitaxel for indicated times. WB performed with indicated antibodies. (E) Indicated cell lines were incubated with paclitaxel for indicated times. Cell lysates were immunoblotted with indicated antibodies. (F) 231 cells were pretreated without or with 1 μM GSK2606414 (a PERK inhibitor) for 2 hr and then incubated with 100 nM paclitaxel for indicated times. WB performed with indicated antibodies. (G) 231 cells were transfected with SLC1A5-V5, treated with DMSO or 100 nM paclitaxel for 24 hr, and then subjected to labeling with (³⁵S) Met/Cys. SLC1A5-V5 was immunopurified and incubated with BNPS-Skatole in vitro for the indicated time points. ³⁵S-SLC1A5 was detected using autoradiography and quantified with the aid of Image J software. (H) 231 cells were transfected with shControl or two different shRNF5s (#1, #2) followed by incubation with 100 nM paclitaxel for 24 hr. WB performed with indicated antibodies. (I) 231 cells transfected with shControl or shRNF5 were incubated with paclitaxel for 24 hr and then treated with cycloheximide (CHX; 50 μg/ml) for indicated times. WB performed with indicated antibodies. (J) 231 cells were transfected with indicated constructs and treated with paclitaxel for 6 hr. Relative ROS levels were determined by DCFDA (2’ 7’-dichlorodihydrofluorescein diacetate) staining. Bars represent means ± s.d. of three independent experiments. See also .

(A) MDA-MB-231 cells transfected with indicated constructs were incubated with 2.5 μg/ml BFA for indicated times. WB performed with indicated antibodies. (B) MDA-MB-231 cells stably expressing the indicated constructs were transfected with a GFP-LC3 expression plasmid and incubated with or without 2.5 μg/ml BFA or with 1 μM rapamycin. Scale bar = 10 μm. (C) Quantification of GFP-LC3 punctae in cells treated as in (B). Bars represent means of ± s.d. of triplicate samples of >50 cells analyzed per sample in three experiments. A two-tailed, unpaired t-test used to calculate significance. (D) MDA-MB-231 cells transfected with indicated constructs were incubated with 2.5 μg/ml BFA for 18 hr, and apoptotic cells were counted using a TUNEL assay. Bars indicate mean values ± s.d. of three experiments. (E) MDA-MB-231 cells transfected with indicated constructs were incubated with 2.5 μg/ml BFA for 24 hr, stained with Annexin-V-EGFP and PI, and analyzed by FACS. Bars indicate mean values ± s.d. of three experiments. (F) MDA-MB-231 cells transfected with indicated constructs were incubated with 100 nM paclitaxel for 48 hr, stained with Annexin-V-EGFP and PI, and analyzed by FACS. Bars indicate mean values ± s.d. of three experiments. See also .

(A, B) H&E staining (A) or IHC staining of vimentin (B) of mammary tumors from MMTV-PyMT;Rnf5^+/+ or MMTV-PyMT;Rnf5^–/– mice. Lower panels show higher magnification images of insets in upper panels. Scale bars: upper panels = 100 μm; lower panels = 50 μm. (C) Left panel: IHC staining of SLC1A5 in mammary tumors from indicated genotypes. Scale bars: upper panels = 100 μm; lower panels = 50 μm. Right panel: Quantification of SLC1A5 staining. Bars represent mean values ± s.d. of groups consisting 10 mice each. (D) Left panel: IHC staining of phospho-S6 ribosomal protein in mammary tumors from indicated genotypes. Scale bars: upper panels = 100 μm; lower panels = 50 μm. Right panel: Quantification of phospho-S6 ribosomal protein staining. Bars represent mean values ± s.d. of groups of 10 mice. (E) Upper panel: MDA-MB-231 BCa cells (5 × 10⁶) stably expressing shRNF5 or shControl were injected into nude mice, and mice were monitored weekly for tumor growth. Animals were subjected to paclitaxel treatment 7 day after injection, as described in Experimental Procedures. Bars represent ± SEM. Lower panel: IHC staining of SLC1A5 or SLC38A2 in tumors generated as indicated in panel E. Scale bars: 50 μm. (F) MDA-MB-231 BCa cells (5 × 10⁶) stably expressing indicated constructs were injected as indicated, monitored and treated as in panel E. Bars represent ± SEM. See also .

(A) Representative immunostaining of a BCa TMA, depicting high and low levels of RNF5, SLC1A5 and SLC38A2 expression. Scale bar = 100 μm. (B) Kaplan-Meier curves comparing disease free survival of BCa patients with tumors expressing high or low levels of SLC1A5 protein (p = 7.5 × 10^–5). (C) Kaplan-Meier curves comparing survival of BCa patients with tumors expressing high or low RNF5 together with high or low levels of SLC1A5 (p = 0.00058). (D) Kaplan-Meier curves comparing survival of BCa patients with tumors expressing high or low RNF5 together with high or low levels of both SLC1A5 and SLC38A2 (SLC* indicates conditions in which both SLC1A5 and SLC38A2 protein levels were either high or low) (p = 0.0021). (E). Heat map depicting expression of SLC1A5 among basal-like, HER2 positive, reactive and luminal BCa in a cohort of 747 samples. See also .