Methotrexate

The following chemicals and materials were obtained from the indicated companies: Dulbecco’s modified Eagle’s medium (DMEM) from Gibco BRL, Grand Island, NY, USA; fetal bovine serum (FBS), benzaldehyde (MW = 106) (purity: >98%), sodium ascorbate (MW = 198), eugenol (MW = 164) (purity: >98%), NaF, D-mannitol, 20% glutaraldehyde solution, dimethylsulfoxide (DMSO) from Wako Pure Chemical, Osaka, Japan; doxorubicin, catalase (EC1.11.1.6, from bovine liver, 41,000 unit/mg protein) from Sigma-Aldrich Inc., St. Louis, MO, USA; mitomycin C from Merck KGaA, Darmstadt, Germany; 5-fluorouracil (5-FU) from Kyowa, Tokyo, Japan; methotrexate from Nacalai Tesque, Inc., Kyoto, Japan; docetaxel from Toronto Research Chemicals, New York, NY, USA; gefitinib from LC Laboratories^®, PKC Pharmaceuticals, Inc., Woburn, MA, USA; SBA (MW = 286) from ChemiScience, Tokyo, Japan; culture plastic dishes and plates (96-well) from Becton Dickinson Labware, Franklin Lakes, NJ, USA. eugenol was dissolved in DMSO at 400 mM before use, and diluted with medium. As a control, cells treated with 0.5% DMSO were used.

Alectinib and LY294002 were purchased from LC Laboratories (Woburn, MA, USA) and Merck Ltd. (Darmstadt, Germany), respectively. Hydrogen peroxide, methotrexate, and catalase were purchased from Nacalai Tesque (Kyoto, Japan). GDC‐0068 and rapamycin were purchased from Cayman Chemical (Ann Arbor, MI, USA) and Toronto Research Chemicals Inc. (Toronto, Canada), respectively. Anti‐β‐actin, anti‐Lamin B, anti‐Akt, anti‐p21, and anti‐p53 antibodies and sodium orthovanadate were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). To detect murine p53, an anti‐p53 antibody was purchased from Merck Millipore (Darmstadt, Germany). An anti‐NPM1 antibody and anti‐DDX21 antibody were obtained from Novus Biologicals (Centennial, CO, USA). An anti‐RPS7 antibody and anti‐RPL23 antibody were purchased from Abgent (San Diego, CA, USA). An anti‐Flag (M2) antibody, anti‐Fibrillarin antibody, and anti‐EBP2 antibody were purchased from were purchased from Sigma‐Aldrich (St. Louis, MO, USA), Abcam (Cambridge, MA, USA), and ProteinTech (Chicago, IL. USA), respectively. An anti‐RPL5 antibody and anti‐RPL11 antibody were purchased from Bethyl Laboratories (Montgomery, TX, USA). Other antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

Soluble CD26 (sCD26) was produced according to the method described previously
[22 (link)]. Briefly, the expression vector RcSRα-26d3-9, which contains a deletion of the coding sequence for amino acids 3–9 of CD26, was transfected into a dihydrofolate reductase deficit Chinese hamster ovary (CHO) cell line, DXB-11 by electroporation, together with pMT-2 providing the dihydrofolate reductase gene. The transfected CHO cells were cultured in serum-free CHO-S-SFM II medium (Invitrogen) supplemented with 1 μM methotrexate (Nacalai Tesque, Kyoto, Japan). The culture supernatant was collected and subjected to affinity chromatography on ADA-Sepharose according to the method described previously
[23 (link)]. Purified sCD26 was denatured in 8 M urea buffer supplemented with 20 mM HEPES and 50 mM dithiothreitol (DTT) by gentle rotation for 8 hours at RT.