A769662

Plasmids expressing de novo purine biosynthetic enzymes conjugated with monomeric enhanced green fluorescent protein (EGFP) or monomeric orange fluorescent protein (OFP) were mostly prepared previously.^{1 (link)} Note that EGFP in this article contains three point mutations, A206K, L221K, and F223R, which prevent potential GFP-mediated oligomerization of tagged proteins.^{36 (link)} We also prepared a new plasmid expressing EGFP-ATIC using the pEGFP-C1 vector (Clontech) to replace the previous clone of GFP-ATIC,^{1 (link)} which was constructed to express GFP_S65T-fusion proteins from the pC1-Neo vector (Promega). The cloned plasmid in this work was confirmed by restriction enzyme digestions and DNA sequencing (GeneWiz). The plasmids expressing shRNA_AMPK (cat# sc-45312-SH) and shRNAControl (cat# sc-108060) were purchased from Santa Cruz.
5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR) and ribonucleotide (ZMP) were obtained from EMD Biosciences. Metformin, phenformin, IMP, AMP, and GMP were obtained from Sigma-Aldrich. A769662 was obtained from Santa Cruz Biotechnology. ATP was obtained from Invitrogen. Cellular staining reagents, including LysoTracker Red DND-99 (cat# L7528), 1,1′-dihexadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiIC₁₆(3); cat# D384), and MitroTrackerOrange CMTMRos (cat# M7510), were obtained from Molecular Probes (Life Technologies).

The U937 cell line was obtained from ATCC in September of 2013. The Kasumi -1 cell line was obtained from ATCC in September 2011. Both cell lines were frozen at low passage (2-3 passages after receipt) in liquid nitrogen and experiments were conducted on cells passaged for a total of no more than 1 month after resuscitation and therefore cells did not need to re-authenticated. AMPKα 1/2 WT and AMPKα1/2 −/− immortalized MEFs were as previously described (36 (link)) and were grown in DMEM supplemented with 10% FBS. Cells were verified for AMPKα knockout by western blot (see Supplemental Fig. S1A). Kasumi-1 cells were cultured in ATCC modified RPMI 1640 supplemented with 20% FBS. U937 cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS). As₂O₃ was purchased from Sigma. PP242 was purchased from Chemdea (Ridgewood, NJ). A769662 was purchased from Santa Cruz Biotechnology and Chemietek. BI-D1870 was purchased from Symansis.