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1 1 dihexadecyl 3 3 3 3 tetramethylindocarbocyanine perchlorate diic16 3

Manufactured by Thermo Fisher Scientific

1,1'-dihexadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiIC16(3)) is a fluorescent dye. It can be used to label the plasma membrane of cells.

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2 protocols using 1 1 dihexadecyl 3 3 3 3 tetramethylindocarbocyanine perchlorate diic16 3

1

Molecular Compound Dissolution and Staining

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Various small molecules were purchased for this study and dissolved in dimethyl sulfoxide (DMSO) solution: GSK2334470 (Sigma), Gö6893 (Calbiochem), PF4708671 (Torcis), Akt inhibitor X (Calbiochem), Akt inhibitor VI (H-AVTDHPDRLWAWEKF-OH; Calbiochem), LY294002 (Calbiochem), and rapamycin (Calbiochem). Cellular staining reagents were purchased from Molecular Probes (Fisher Scientific), which included 1,1’-dihexadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate (DiIC16(3); Cat# D384), and MitoTrackerOrange CMTMRos (Cat# M7510).
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2

De Novo Purine Biosynthesis Enzymes

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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 GFPS65T-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 shRNAAMPK (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).
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