[U- 13 C5] glutamine. Glutamate. Acetyl-coA. Citrate. Citrate. Malate. Malate. Isocitrate OXIDATIVE METABOLISM. Oxaloacetate CO2.

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1 Supplementary Figures a. Relative mrna levels Supplementary Figure 1 (Christofk) LAT1 Fumarate Succinate Palmitate Acetyl-coA Oxaloacetate OXIDATIVE METABOLISM α-ketoglutarate Acetyl-coA α-ketoglutarate Oxaloacetate tamate e artate Fumarate [U- 13 C5] glutamine MID (U tracer) c. % 13 C labeled (U tracer) M5 tamate M5 αkg Succinate Fumarate d. e. AD D68A Relative mrna levels artate C. 0 M0 M1 M2 M3 M5 M6 0.0 IDH1 IDH2 NNT PDK1 GOT2 Supplementary Figure 1: (a) qrt-pcr was performed on RNA collected from MCF10A cells 8 hours post infection with or AD ORF1 D68A virus. Relative levels of and LAT1 are shown. For (b)-(e), NHBE cells were labeled with U- 13 C-glutamine (U tracer) and mock infected or infected with or AD ORF1 D68A at an MOI of hours post infection, intracellular metabolites were extracted and analyzed by LC-MS/MS. (b) Schematic tracing the fate of 13 C atoms from U- 13 C 5 -glutamine in oxidative metabolism in the TCA cycle. (c) Percentage of 13 C-labeled isotopomers of intermediates in oxidative TCA cycle metabolism. (d) mass isotopomer distribution (MID) resulting from U- 13 C-glutamine labeling. (e) qrt-pcr was performed as described in (a), and relative levels of transcripts involved in reductive carboxylation are shown.

2 Supplementary Figure 2 (Christofk) Pyr OAA Mal Fum AcCoA Pyr PDH Asn PDK1 NADP+ NADPH α-kg IDH2 GOT2 OAA NADP+ NADPH Pro NNT KEY: Metabolite increased labeling from U-13C- in but not AD ORF1 D68A infected cells Metabolite difficult to measure via current LC-MS method Enzyme transcript level elevated in but not AD ORF1 D68A infected cells NEAA EAA LAT1 Supplementary Figure 2: Schematic of reductive glutamine carboxylation and glutamine-associated amino acid metabolism events summarizing the MYC-dependent changes observed in adenovirus-infected cells.

3 Supplementary Figure 3 (Christofk) 2" a. Relative mrna Levels 1.5" 1" 0.5" GFPT1 NAGK KEY: Metabolite level elevated in but not AD ORF1 D68 infected cells Metabolite level elevated and increased M2 labeling from U-13C- in but not AD ORF1 D68A infected cells Enzyme transcript level elevated in but not AD ORF1 D68A infected cells cose HK2 cose-6-phosphate Fructose-6-Phosphate GFAT1 cosamine-6-phosphate Acetyl CoA CoA GlcNAc-6-Phosphate GlcNAc-1-Phosphate UDP-GlcNAc NAGK Pentose Phosphate Pathway Glycolysis GlcNAc HEXOSAMINE BIOSYNTHESIS N-glycosylation initiation N-glycan branching O-GlcNAc modification Supplementary Figure 3: (a) qrt-pcr was performed on RNA collected from MCF10A cells 8 hours post infection with or AD ORF1 D68A virus. Relative levels of GFPT1 (glutamine fructose-6-phosphate amidotransferase 1) and NAGK (N-acetylglucosamine kinase) are shown. (b) Schematic of the hexosamine biosynthesis pathway summarizing the MYC-dependent changes observed in adenovirus-infected cells.

4 Supplementary Figure 4 (Christofk) a. ODC1 FPKM values FPKM values 5" 4" 3" 2" 1" PRODH Supplementary Figure 4: Preliminary RNA-sequencing data comparing uninfected NHBE cells () versus cells infected for 8 hours with (). The fragments per kilobase of transcript per million mapped (FPKM) values indicate that RNA levels of (a) ornithine decarboxylase (ODC1) and (b) proline dehydrogenase (PRODH) are increased at 8 hours post- infection.

5 Supplementary Figure 5 (Christofk) Mock ADWT 48h ADWT 24h a. Figure 1b MYC MYC Figure 1d GLS GLS1 shgls1-2 shgls1-1 shgls1-2 c. shgls1-1 Figure 3b Supplementary Figure 5: Uncropped Western Blot images presented in this manuscript. Figure labeling corresponds to the figure in the main manuscript.

Supplementary Material. Contents include:

Supplementary Material. Contents include: Supplementary Material Contents include: 1. Supplementary Figures (p. 2-7) 2. Supplementary Figure Legends (p. 8-9) 3. Supplementary Tables (p. 10-12) 4. Supplementary Table Legends (p. 13) 1 Wellen_FigS1