Fig 2: ALK signaling upregulates amino acid transporter systems.Gene oncology GSEA analysis of RNA-seq datasets. A NB tumors from either Alk-F1178S;Th-MYCN mice or Rosa26_Alkal2;Th-MYCN mice compared to Th-MYCN mice. B NB1 cells treated with either ALKAL2 or/and lorlatinib for 24 h. C CLB-BAR cells treated with lorlatinib for 24 h compared to DMSO treated control. D Heat map indicating biotinylation of SLC1A5, SLC3A2, SLC6A15, SLC38A1 in SK-N-AS.ALK-BirA* expressing cells compared to BirA* controls. Columns represent three biological replicates, data from [41]. Immunoblotting for anti-pALK (Y1278), SLC1A5, SLC3A2, SLC7A5, SLC7A11, SLC38A1 and GAPDH in NB1 cells treated with ALKAL2 (1 µg/ml) and lorlatinib (0 or 30 nM) for 24 h (E) and CLB-BAR cells treated with lorlatinib (0, 30 nM) for 24 h (F). G Schematic visualization of the regulation of SLC3A2 downstream of ALK in NB cells (created with BioRender.com). Immunoblot experiments were performed in biologically independent triplicates. Quantifications were performed with ImageJ and analyzed by Student t test (unpaired, two-tailed). p values are indicated (****P < 0.0001, ***0.0001 < P < 0.001, **0.001 < P < 0.01, *0.01 < P < 0.05, ns P ≥ 0.05); exact P values are shown in Supplementary Fig. 5 and Supplementary Table 1.

Fig 3: Effects of inhibiting the expression of SLC38A1 on ESCC cell proliferation and migration. (A) SLC38A1 was upregulated within KYSE-150 and Eca109 cells relative to HEECs by the qRT-PCR assay and western blot analysis. (B) The expression of SLC38A1 was downregulated in KYSE-150 and Eca109 cells by siRNAs. (C–E) KYSE-150 and Eca109 cell proliferation after anti-SLC38A1 siRNA transfection was measured using CCK-8 assays (C, D) and colony formation assays (E). (F) Transwell assays for ESCC cell migration (scale bars: 100 μm).

Fig 4: Development of a cell line to characterize SNAT2 activity.A and B, SNAT2 immunoblot in HY15549 iSNAT2 after withdrawal of doxycycline (dox) over the course of 24 h. Densitometry analysis, relative to a parallel actin immunoblot, suggests an SNAT2 half-life of approximately 6 h after dox withdrawal. C, SNAT2 and sodium/potassium (N/K)-ATPase immunoblots in whole cell lysate (WCL), postnuclear supernatant (PNS), total membrane (TM), and plasma membrane (PM) samples isolated from HY15549 iSNAT2 cells cultured with or without dox (0.5 μg/ml) for 24 h. An equal amount of protein (20 μg) was loaded for each sample, which was confirmed using a Ponceau S stain. D, SNAT1, SNAT2, and N/K-ATPase immunoblots in HY15549 iSNAT2 EV or + SNAT1 cell lines cultured with or without dox for 24 h. Each immunoblot is representative of n ≥ 1 independent experiments. EV, empty vector; SNAT2, sodium-coupled neutral amino acid transporter 2.

Fig 5: MeAIB uptake using GC–MS quantifies SNAT1 or SNAT2 activity.A, schematic showing the sodium-dependent uptake of MeAIB by system A transporters, as quantified by GC–MS. B, extracted ion chromatogram (XIC) of representative GC–MS samples for 174 m/z, corresponding to the main fragment ion of tertbutyldimethylsilylated MeAIB. C, MeAIB uptake in HY15549 iSNAT2 cells cultured with (+SNAT2) or without (SNAT2-KO) doxycycline (dox). D, MeAIB uptake in HY15549 iSNAT2 cells cultured with dox performed in PBS with or without sodium. E and F, MeAIB uptake in HY15549 iSNAT2 EV (E) or +SNAT1 (F) cells cultured with or without dox. C–F, all MeAIB uptake assays were performed 24 h after plating with or without dox to allow for SNAT2 expression to extinguish, in the case of no supplemented dox. Data in (C–F) are mean ± SD of n = 3 independent experiments; the Michaelis–Menten constant (KM) was estimated using a nonlinear fit in Prism. MeAIB, α-(methylamino)-isobutyric acid; SNAT1/2, sodium-coupled neutral amino acid transporter 1/2.