Folate

l-Methionine, d,l-homocysteine, vitamin B12, and folate were purchased from Sigma-Aldrich (St. Louis, MO, USA). Rabbit monoclonal anti-p-eIF2α and rabbit polyclonal anti-β-Tubulin were purchased from Cell Signaling Technologies (Danvers, MA, USA). Rabbit polyclonal anti-phospho- IRE1α was purchased from Novus Biologicals (Littleton, CO, USA). Rabbit polyclonal anti-t-IRE1α, rabbit polyclonal anti-GRP78, mouse monoclonal anti-CHOP, and rabbit polyclonal anti-homocysteine were purchased from Abcam (Cambridge, MA, USA). Annexin V-FITC Kit was purchased from MBL (Nagoya, Japan).

Mouse embryonic stem cells (ESCs; Sv/129) were obtained from Xuanwu Hospital (Beijing, China), and maintained as mitotically inactivated primary mouse embryonic fibroblasts prior to culture under feeder-free conditions. ESCs were seeded onto culture dishes coated with 0.2% gelatin (Sigma-Aldrich, St Louis, MO, USA) and maintained in folate-free Dulbecco’s modified Eagle’s medium (Sigma-Aldrich, St Louis, MO, USA) supplemented with 0.1 mMb-mercaptoethanol, nonessential amino acids, 2 mM glutamate, 15% fetal bovine serum (all purchased from Invitrogen Life Sciences, Carlsbad, CA, USA), 4 mg/l folate (Sigma-Aldrich, St Louis, MO, USA) and 1000 U/ml leukemia inhibitory factor (Millipore, Billerica, MA, USA). Cells were passaged every 3 days. After three passages under folate-normal (4 mg/l folate) conditions, mouse ESCs were divided into two groups; cultured in folate-normal dedium (FN) or medium without folate (folate-free group, defined as FF). Mouse embryonic bodies (EBs) were differentiated from ESCs after removal of leukemia inhibitory factor [47 (link)]. Cells were maintained at 37°C in a humidified atmosphere with 5% CO₂. Medium changes were performed daily.

Folate, amino acids, nucleosides, nucleotides and other chemical compounds were purchased from Sigma-Aldrich (St. Louis, MO, USA). Minimal essential medium/alpha modified (αMEM) without ribosides, ribotides, deoxyriboside, deoxyribotides, glycine, serine and Folate was specially ordered and formulated by Invitogen and JRH (Lenexa, KS, USA). Fetal bovine serum (FBS) was from Biological Industries (Kibbutz, Israel). Penicillin, streptomycin, fungizone, trypsin and trypan blue were from GIBCO Laboratories (Grand Island, NY, USA).
Antibodies specific for heavy subunit of γ-glutamylcysteine synthetase (γ-GCS_h), COX-2, Bcl-2, GRP78, ATF4, ATF6α, SP-1 (Santa Cruz Biotechnology, california, USA), Survivin, pro-caspase 3 (Cell Signaling Technology, Danvers, MA, USA), PARP-1, β-catenin (Epitomics, california, USA), and β-actin (Sigma-Aldrich, St. Louis, MO, USA) were obtained.

Linear ssDNA template, T7 promoter primer, 3′-cholesterol-modified DNA conjugate (DNA–Chol), 5′-amine-modified DNA fragment for preparation of FA–DNA, 3′-Cy5-labelled and 5′-amine-modified DNA fragment for preparation of FA–DNA-Cy5, primers for qRT–PCR, scrambled sense siRNA, scrambled antisense siRNA and monomeric siRNA were all synthesized from Bioneer Co. (Daejeon, Korea). All DNA sequences are summarized in Supplementary Table 1. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), Sulfo-NHS and folate were purchased from Sigma (St Louis, MO, USA). Lipofectamine2000 was purchased from Invitrogen (USA). Cell culture media were obtained from WelGENE (Korea). The RNA mfold program (available in http://mfold.rna.albany.edu/?q=mfold/RNA-Folding-Form) was used to predict the RNA secondary structures45 (link). Female BALB/c nude mice and female C57BL/6J mice were purchased from Orient Bio Inc. (Korea).