Nec854010uc

For methionine uptake assays, cells were cultured and labeled as described above for the protein synthesis assay, were washed three times in cold PBS, and lysed in Triton lysis buffer. For cystine uptake assays, cells were treated the same but labeled for the final 10 min with medium containing 0.1 µCi L-[1, 2, 1', 2'-¹⁴C]-Cystine (PerkinElmer, NEC854010UC) and washed three times in ice-cold PBS containing cold cystine (1 mM), prior to lysis in Triton lysis buffer. Whole-cell radiolabel incorporation was quantified with a Beckman LS6500 scintillation counter. Cells from identically treated parallel plates were counted using a Beckman Z1-Coulter Counter to normalize uptake measurements to cell number.

Reagents used in this work include sorafenib (sc-220125, Santa Cruz), sulfasalazine (S0883, VWR), erastin (329600, Sigma-Aldrich), β-ME (31350010, ThermoFisher), liproxstatin-1 (Lip-1) (S7699, Selleck Chemicals), deferiprone (DFP) (379409, Sigma-Aldrich), (1S,3R)-RSL3 (RSL3) (Cay19288, Cayman Chemical), iFSP1 (8009-2626, ChemDiv) [15 (link)], and [¹⁴C]cystine (NEC854010UC, PerkinElmer).

The influence of ASCTs, SNATs, and LAT1 transporters on the uptake of ¹⁴C-cysteine was measured. Cells (2.5 × 10⁵) were seeded into 35 mm dishes and after 24 h, culture media were removed and cells were washed with Hank’s Balanced Salt Solution (HBSS: 125 mM NaCl, 4.8 mM KCl, 1.2 mM MgSO₄, 1.2 mM NaH₂PO₄, 1.3 mM CaCl₂, 5.6 mM glucose, and 25 mM HEPES), incubated in 1 mL of prewarmed HBSS at 37 °C for 5 min. Following, the cells were incubate at room temperature for 30 min in 1 mL HBSS supplemented with L-[1,2,1′,2′-¹⁴C]cysteine (0.2μCie/mL; PerkinElmer Ref: NEC854010UC) + 100 μM β-mercaptoethanol, in order to keep cysteine in its reduced form) and 50 μM cold cystine in the presence or not of 10 mM L-alanine (ASCTs transporters competitive inhibitor), 10 mM L-Leucine (LAT1 transporter competitive inhibitor) or 10 mM N-methylaminoisobutyric acid (MeAIB, Merck, Darmstadt, Germany, SNATs transporters inhibitor). Subsequently, cells were washed 3 times with HBSS solution containing the specific carriers’ inhibitors and lysed with 1 mL of 1 M NaOH, following addition of 12 mL of Emulsifier-Safe cocktail (PerkinElmer, Waltham, MA, USA). Radioactivity was measured using a β–scintillation counter. Relative L-[¹⁴C-cysteine] uptake was normalized by protein content. L-alanine (A7627), L-leucine (L8000), and MeAIB (M2383) were purchased from Sigma-Aldrich.