Methotrexate mtx

Methotrexate (MTX) (Sigma Aldrich, St. Louis, MO, USA) was dissolved in PBS (1 mg/10 mL) and mixed with bi-HNTs (200 mg) and keep stirring for 12 h, then, the mixture was vacuumed for 24 h. After centrifugation, the supernatant liquid was collected and stored at −20 °C for drug loading efficiency determination, and the bottom deposited bi-HNTs were washed by PBS for 3 times and air-dried, the final product was collected for further study.

BT-474, S-180, MCF10A, MCF7, and NMUMG cell lines were purchased from ATCC and cultured as previously described^{26 (link),47 (link)}. NT2196 is a stable cell line corresponding to immortalized NMUMG cells that were transformed with an oncogenic form of Neu (ErbB2), which was cultured as previously described^{48 (link)}. AMPK-null MEFs and eμ-Myc B cell lymphoma shCTRL/shAMPK cell lines were generous gifts from Dr. Russell Jones’ laboratory, and were cultured as previously described^{31 (link),45 (link),49 (link)}. The eμ-Myc B cell lymphoma cell line was engineered using the plasmid MSCV p2GM AMPK alpha2hp1 alpha1hp1 (Addgene plasmid # 89492; http://n2t.net/addgene:89492; RRID:Addgene_89492). Methotrexate (MTX) (dissolved in DMSO) and phenformin (dissolved in water) was purchased from Sigma. All LC/MS grade solvents and salts were purchased from Fisher (Ottawa, Ontario Canada: water, acetonitrile (ACN), Methanol (MeOH), formic acid, and ammonium acetate. The authentic metabolite standards were purchase from Sigma-Aldrich Co. (Oakville, Ontario, Canada).

HeLa and Caco-2 cells (passages 24–26) were regularly passaged in minimal essential medium α-modification (Hyclone) supplemented with 10% FBS (Hyclone) and 1% penicillin/streptomycin (Corning). For experiments to determine concentration of an inhibitor where the response is reduced by half (IC₅₀), of antifolates, cells were cultured in defined minimal essential medium α-modification that lacks ribonucleotides, deoxyribonucleotides, folate, pyridoxine, methionine, serine, and glycine and supplemented with 10% (vol:vol) dialyzed, charcoal-treated FBS (Hyclone), 200 µM methionine, 1 mg pyridoxine/L, and 2 µM folic acid. HeLa cells were seeded into 96-well plates at densities of 2000 cells/well, and Caco-2 cells were plated at 5000 cells/well. Immediately after seeding, cells were treated with 50 µM deoxyuridine or 50 µM uridine, and 6 concentrations of 5-fluorouracil (5-FU; 0–1000 µM; Sigma) or methotrexate (MTX; 0–0.5 µM; Sigma) with n = 10 wells per treatment condition and cultured for 96–120 h. Cell viability was measured by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and IC₅₀ values were calculated with the use of Prism software as previously described (16 (link)).