Gsh mee

The validated cell lines HEK293T, U-373 MG and U-87 MG were maintained in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum. Human glioblastoma explants, GB1, GB2, GB3 and GB4, were kindly supplied by Dr Marta Izquierdo (Centro de Biología Molecular “Severo Ochoa” - Autonomous University of Madrid). Most experiments with these explants were performed with GB1 and GB3 because they exhibit the highest proliferative rates. All experiments were conducted under neurosphere culture conditions as described previously [24 ]. Inmortalized human neural stem cells derived from ventral mesencephalon of fetal brain (ReNcell) were plated onto Corning® Matrigel® hESC-Qualified Matrix (CORNING) and maintained in Neurobasal medium (Gibco) containing 2% B27 Supplement (Gibco) (v/v), 20 ng/ml recombinant human EGF (Peprotech), 20 ng/ml recombinant human basic FGF (Peprotech), 100 U/ml Penicillin/Streptomycin (Life Technologies) and 1% Amphotericin b solution (Lonza) in 5% CO₂ at 37 °C conditions. Sulforaphane (SFN) and GSH-MEE were purchased from Sigma-Aldrich. Limiting dilution assays were performed essentially as described in Ref. [25 (link)]. The final data and the statistical significances were calculated using the Extreme Limiting Dilution Analysis (ELDA) software (http://bioinf.wehi.edu.au/software/limdil/index.html) [25 (link)].

LMPTP inhibitor Compd. 23 was generated as described (14 (link)) and formulated in rodent chow at 0.1% (w/w) by Research Diets. CRISPR-Cas9 plasmids were purchased from ATUM. Pierce d-Luciferin monopotassium salt was purchased from Thermo Fisher Scientific and reconstituted using phosphate-buffered saline (PBS). For in vitro experiments, docetaxel and cabazitaxel were purchased from Selleckchem and reconstituted in sterile dimethyl sulfoxide (DMSO). The anti–phospho–eIF2-Ser⁵¹ (#3398), anti-eIF2 (#9722), anti-NRF2 (#12721), anti-ATF4 (#11815), anti–glyceraldehyde 3-phosphate dehydrogenase (#5174), anti–phospho-H2AX (#9718), anti-H2AX (#2595), and anti-pTyr (pY1000; #8954) antibodies were purchased from Cell Signaling Technology (CST). The rabbit anti-LMPTP antibody was described in (52 (link)). The mouse anti-LMPTP antibody (#sc-100343) was purchased from Santa Cruz Biotechnology. The anti-GSS antibodies were purchased from Thermo Fisher Scientific (#PA5-89891) or Santa Cruz Biotechnology (#sc-166882). Anti-rabbit (#NA934-1ML) and anti-mouse (#NA931-1ML) secondary antibodies were purchased from Thermo Fisher Scientific. TrueBlot anti-rabbit (#RL18-8816-31) and anti-mouse (#RL18-8817-31) secondary antibodies were purchased from Rockland. GSH-MEE (#353905) was purchased from Sigma-Aldrich. Unless otherwise specified, chemicals and other reagents were purchased from Sigma-Aldrich.

To study the effect of oxidative stress on expression of OGC and DIC, the cells were exposed to H₂O₂ at varying doses (50, 100, 200, 300 μM) for 24 hours, and varying durations (2, 4, 6, 8, 24 hours) with 200 μM H₂O₂. To identify dose and time-dependent inhibition of OGC and DIC expression by chemical inhibitors, cells were incubated with phenylsuccinic acid (PS) and butylmalonic acid (BM; Sigma-Aldrich Corp., St. Louis, MO, USA) in varying doses (2, 5, 10 mM) for 24 hours, and varying durations (2, 4, 6, 8, 24 hours) with a single 5 mM dose of either PS or BM, respectively. Cells were also treated with 5 mM PS or BM, in the presence or absence of 2 mM GSH-MEE (Sigma-Aldrich Corp.) for 24 hours. To identify the effect of competitive inhibitors of the two transporters, cells were treated with a 5 mM dose of either dimethyl 2-oxoglutarate or diethyl malate for 24 hours. All inhibition studies were performed with RPE cells in serum-free medium containing 0.1% dimethyl sulfoxide.

For the induction of autophagy by amino acid starvation cells were washed three times with PBS and cultured in Earle’s Balanced Salt Solution (EBSS, Thermo Fisher) for 4 or 24 h at 37°C. The PIKfyve kinase inhibitor Apilimod (Selleckchem) was used at 20 nm for 60 min at 37°C. The specific inhibitor of vacuolar-type H⁺-ATPase Bafilomycin A1 (Merck) was used at 200 nm for 30 min or 4 h at 37°C. The antioxidant glutathione reduced ethyl ester (GSH-MEE, Sigma-Aldrich) was used at 1 mm for 24 h at 37°C.