D 3h aspartate

SiO₂-NPs ranging from 10 to 20 nm in size, dimethyl sulfoxide (DMSO) (#M81802), and 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl-2H-tetrazolium bromide (MTT; # M2128) were obtained from Sigma–Aldrich (St. Louis, MO, USA). [³H]-D-aspartate was purchased from PerkinElmer (Boston, MA). Cell culture medium was from Thermo Fisher Scientific (Carlsbad, CA), and plasticware was purchased from Corning (New York, NY).

System x_c⁻-specific ¹⁴C-L-cystine (PerkinElmer; Waltham, MA) and system X_AG⁻-mediated ³H-D-aspartate (PerkinElmer; Waltham, MA) uptake was performed as previously described (Fogal et al., 2007 (link)). Cultures were washed into HCSS (3 × 750 µl) and allowed to equilibrate for 10 min (25℃). For cystine uptake, cells were incubated in HCSS containing 3 µM ¹⁴C-L-cystine (1 µCi/ml), 27 µM unlabeled cystine, 1 mM D-aspartate, and 0.5 mM acivicin (Enzo Life Sciences; Farmingdale, NY). D-aspartate and acivicin were included in the uptake buffer to block system X_AG⁻ and γ-glutamyltranspeptidase, respectively. Uptake was terminated after 30 min by washing in ice-cold PBS (3 × 750 µl). For D-aspartate uptake, cells were incubated in HCSS containing 0.1 µCi/ml ³H-D-aspartate and 50 µM unlabeled D-aspartate (25℃) for 5 min and uptake terminated by washing cells with an ice-cold Na⁺-free choline stop buffer containing in mM: 116 choline chloride, 0.8 MgSO₄, 1 KH₂PO₄, 10 HEPES, 5 KOH, 10 glucose, 0.9 CaCl₂, and 5 nonradioactive D-aspartate.
Cells were lysed with warm 0.5% SDS and accumulated radioactivity estimated using a liquid scintillation counter. Readings of counts per minute from experimental conditions were corrected back to the original volume of lysate, and the picomoles of cystine and aspartate transported per minute were calculated as described (Fogal et al., 2007 (link)).

On day 21–25, primary mixed cultures (propagated on 18-mm-cover slips in 12-well-plates) were washed 3 × with 0.5 mL Hepes Krebs-Ringer buffer. Aspartate uptake was allowed for 5 min at RT by incubation in 0.5 mL PBS containing 200 μM D-aspartate and [³H]-D-aspartate 0.15 μCi/well (PerkinElmer Inc, Waltham, MA, USA). Cover slips were rinsed 3 × with 0.5 mL cold Hepes Krebs-Ringer buffer, and subsequently lysed in 0.5 mL 0.1 M NaOH. The protein content in all lysates was determined using a method adapted from Bradford25 (link) (RC DC protein assay kits, 500-0113 and 500-0114, Bio-Rad Laboratories Inc). The content of [³H]-D-aspartate in 0.45 mL lysate was mixed with 5 mL of scintillator (Ready Safe, 141349, Beckman Coulter Inc, Brea, CA, USA) and quantified by liquid scintillation spectrometry using a WinSpectral 1414 scintillation counter (Wallac, Turku, Finland). The counts per minute (CPM) were normalized to the protein amount (μg), see Supplementary Table 5. In each experiment, the average [³H]-D-aspartate uptake (CPM/μg protein) for WT samples was set to 100% and, subsequently, all samples were normalized (in %) relative to WT. The normalized data ([³H]-D-aspartate uptake as % of WT) were shown as means ± s.e.m. and analyzed using one-way ANOVA, see Supplementary Table 5.