Dialysed fbs

All SILAC reagents were sourced from Thermo Fisher; except for dialysed FBS (Sigma). Lentivirally transduced GFP and GFP-SNX3 RPE1 cells were grown in SILAC DMEM for at least 6 passages to achieve full labelling. GFP-expressing control cells were grown in unlabelled medium containing regular arginine and lysine (R0, K0). GFP-SNX3-expressing cells were grown in medium containing ¹³C₆-arginine and 4,4,5,5-D4-lysine (R6, K4). GFP was precipitated with GFP-trap beads (Chromotek) for 60 min. The beads were pooled, proteins were eluted in sample buffer, separated on Nupage 4–12% precast gels (Invitrogen) and subjected to LC–MS/MS analysis on an Orbitrap Velos (Thermo) mass spectrometer^{40 (link)–43 (link)}.

Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), dialysed FBS, penicillin/streptomycin, glutamine, metformin hydrochloride and hexachlorophene (Hex) were purchased from Sigma Aldrich (St. Louis, MO). A769662 was purchased from Abcam (Cambridge, UK). SBI0206965 (6965) was purchased from Cayman chemical (Ann Arbor, MI).
HEK293, T47D, Huh7, MCF7, A549, HACAT, MCF10A, LNCAP, HT29, HCT116 and SH-SY5Y mammalian cell lines were cultured in DMEM with high glucose (4.5 g/L) supplemented with 10% FBS, 1% penicillin/streptomycin and 8 mM glutamine. For growth assays and cell lysates collected for western blot, cells were washed with Dulbecco’s phosphate buffered saline (DPBS) prior to culture with normal medium (Control (+gln); DMEM high glucose, 10% dialysed FBS, 1% penicillin/streptomycin and 8 mM glutamine) or glutamine depleted media with (−gln + NH₄Cl) or without 0.8 mM ammonium chloride (−gln). Additionally, cells cultured in glutamine depleted medium with or without ammonium chloride were treated with or without the AMPK activator, A769662 (100 µM or 200 µM) or metformin (5 mM), GDH inhibitor, hex (2.5 µM or 5 µM) or the Ulk1 inhibitor, 6965 (10 µM). Control cells were cultured in glutamine depleted medium with or without ammonium chloride and dimethyl sulfoxide (DMSO) or water as vehicle control.