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1 Supplementary Material Contents include: 1. Supplementary Figures (p. 2-7) 2. Supplementary Figure Legends (p. 8-9) 3. Supplementary Tables (p ) 4. Supplementary Table Legends (p. 13) 1
2 Wellen_FigS1 A IL-3Ra mrna Relative expression B Glc +Glc +GlcNAc PNGase F - + Glycosylated 62 Unglycosylated 49 IL-3Ra 2
3 Wellen_FigS2 Primary Bone Marrow Cells M-NFS-60 cells (murine myeloblastic) fl *** Arbitrary Units *** 300 -Glc -Glc +GlcNAc 400 -Glc -Glc +GlcNAc 3
4 Wellen_FigS3 A. B. C. A. Cell Size B. 900 fl Cell number * 10 5 Cell Number mm Ammonia Production 4
5 Wellen_FigS4 A. B. % of glucose+ IL-3Ra Surface Expression 120 Glc- 100 Glc- GlcNAc UT CSN DJ Tun Glucose + GlcNAc pstat5 Stat5 ps6 S6 peif2a eif2a CSN Tun C. fl Cell Size Ctrl CSN DJ Tun Glc- Glc-GlcNAc+ D. Arbitrary Units IL-3Ra Surface expression *** Mgat5-wt Mgat5(L188R) 5
6 6 Wellen_FigS5
7 Wellen_FigS6 Ctrl BHA NAC GlcNAc pstat5 (Tyr 694) Stat5 7
8 Supplementary Figures Figure S1. A) IL-3-dependent cells were withdrawn from glucose for 24 hours, then treated with either 15 mm glucose, 15 mm GlcNAc, or left untreated for an additional 24 hours. RNA was isolated from triplicate wells and IL-3R gene expression normalized to that of 18S rrna. Results represent mean +/- s.d. B) Membrane protein fractions were isolated from IL-3-dependent cells after 48 hours culture in the presence of glucose. Half of each sample was treated with the deglycosylating enzyme PNGase F and then treated and untreated samples analyzed by Western blot. Figure S2. M-NSF-60 cells were withdrawn from glucose in the presence or absence of 15 mm GlcNAc for 2 days. Cell size in PI negative cells was determined by flow cytometry (mean +/- s.d. of triplicate samples). Primary bone marrow cells were withdrawn from glucose in the presence or absence of 15 mm GlcNAc for 4 days and cell size was determined in fl (mean +/- s.d. of triplicate samples; ***, p<0.0005). Figure S3. IL-3-dependent bax-/-bak-/- cells were withdrawn from glucose for 3 days in the presence of 0.5 mm or 1 mm glucosamine or 15 mm GlcNAc. Cell size (A), cell number (B), and ammonia production (C) were determined (mean +/- s.d. of triplicates). Figure S4. IL-3R surface expression is regulated by GlcNAc in a glycosylation-dependent manner. A) IL-3R surface expression determined by FACS after 2 days of incubation in glucose-free media, +/- 15 mm GlcNAc, +/- inhibitors (100 M castanospermine (CSN), 100 M deoxynojirimycin (DJ), 3 g/ml tunicamycin (tun)). Results represent mean +/ s.d. of 3 independent experiments. B) Western blots after 24 hours glucose starvation, followed by 24 hours GlcNAc addition in the presence or absence of 100 M CSN or 3 g/ml tunicamycin. Result is representative of 2 independent experiments. C) Cell size (mean +/- s.d. of triplicate wells) after 24 hours glucose starvation, followed by 24 hours GlcNAc addition, in the presence or absence of indicated inhibitors. D). Wild type Mgat5 or a mutant Mgat5 (L188R) that 8
9 fails to localize to the Golgi were stably expressed in IL-3-dependent cells. Expression and Mgat5 activity were confirmed by enzymatic assays (data not shown). IL-3R surface expression was analyzed by FACS following culture in IL-3-containing complete medium (mean +/- s.d. of triplicates, *** p<0.0005)). Figure S5. The hexosamine pathway coordinates glucose and glutamine metabolism through regulation of IL-3R surface expression in hematopoietic cells. The data suggest that glucose flux into the hexosamine pathway enables proper glycosylation and surface expression of IL-3R. IL-3 binds to IL-3R, promoting the formation of an oligomeric complex containing IL-3, IL-3R, and IL-3R c and stimulating IL-3-dependent signaling. IL-3 stimulates uptake of glutamine, in a Jak-dependent manner. Glutamine fuels cell growth through its utilization in the TCA cycle for production of ATP and precursors for fatty acid and non-essential amino acid synthesis, as well as by promoting uptake of essential amino acids to allow mtor activity and protein synthesis. In the absence of glucose, IL-3R does not express at the cell surface, inhibiting IL-3-dependent signaling, glutamine consumption, and cell growth. Figure S6. Antioxidant treatment blocks Stat5 phosphorylation in the presence of GlcNAc. Cells were withdrawn from glucose for 24 hours and then treated with GlcNAc in the presence or absence of 100 M Butylated hydroxyanisole (BHA) or 10 mm N-acetyl-L-cysteine (NAC) for an additional 24 hours. IL-3 was present in all conditions. Cells were harvested and signaling analyzed by Western blot. 9
10 Table S1: Total metabolites (Labeled + Unlabeled) Hexosamine GlcNAc-P 1h h h UDP-GlcNAc 1h h h Glycolysis Lactate 1h h h Hexose-P 1h h h PG 1h h h FBP 1h h h Pentose Phosphate Ribose-P 1h h h PRPP 1h h h TCA Succinate 1h h h Malate 1h h h Ketoglutarate 1h h h Citrate 1h h h
11 Other Glycerol-P 1h h h Glutamate 1h h h Reduced glutatione 1h h h Oxidized glutatione 1h h h N-acetyl-glutamate 1h h h Ornithine 1h h h
12 Table S2. Labeled metabolites Hexosamine no label 13 C-Glucose 13 C-GlcNAc GlcNAc-P 1h h h UDP-GlcNAc 1h h h Glycolysis Lactate 1h h h Hexose-P 1h h h PG 1h h h FBP 1h h h Pentose Phosphate Ribose-P 1h h h PRPP 1h h h TCA Malate 1h h h Ketoglutarate 1h h h Citrate 1h h h Other Glycerol-P 1h h h Glutamate 1h h h
13 Supplementary Tables Table S1. IL-3-dependent cells were starved of glucose for 24 hours. Either 15 mm 13 C 6 -glucose, 15 mm 13 C 6 -GlcNAc, or water was added to cells. Cells were harvested and metabolites extracted at 1, 24, and 72 hours after metabolite addition. Metabolites were analyzed by LC-MS/MS. Shown are the sums of labeled + unlabeled metabolites in each sample, equal to total pool size. Table S2. IL-3-dependent cells were starved of glucose for 24 hours. Either 15 mm 13 C 6 -glucose, 15 mm 13 C 6 -GlcNAc, or water was added to cells. Cells were harvested and metabolites extracted at 1, 24, and 72 hours after metabolite addition. Metabolites were analyzed by LC-MS/MS. Shown is the total of labeled metabolites in each pool (sum of each labeled species). Note that non-zero values were obtained for some metabolites, even when no 13 C label was introduced; these readings represent background or natural abundance. 13
[U- 13 C5] glutamine. Glutamate. Acetyl-coA. Citrate. Citrate. Malate. Malate. Isocitrate OXIDATIVE METABOLISM. Oxaloacetate CO2.
Supplementary Figures a. Relative mrna levels Supplementary Figure 1 (Christofk) 3.0 2.5 2.0 1.5 1.0 0.5 0.0 LAT1 Fumarate Succinate Palmitate Acetyl-coA Oxaloacetate OXIDATIVE METABOLISM α-ketoglutarate
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Supplemental Figure 1 Metabolic flux with [U- C]Lactate - [ 12 C]Glutamine in primary hepatocytes a b c d e [ C 3 ]Pyruvate [ C 3 ]Malate [ C 3 ]Aspartate [ C 3 ]Citrate [ C 2 ] -Ketoglutarate f g h [
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