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 A. 1.6 1.4 IL-3Ra mrna Relative expression 1.2 1 0.8 0.6 0.4 B. 0.2 0 -Glc +Glc +GlcNAc PNGase F - + Glycosylated 62 Unglycosylated 49 IL-3Ra 2
Wellen_FigS2 Primary Bone Marrow Cells M-NFS-60 cells (murine myeloblastic) fl 800 700 600 500 400 *** Arbitrary Units 800 700 600 500 *** 300 -Glc -Glc +GlcNAc 400 -Glc -Glc +GlcNAc 3
Wellen_FigS3 A. B. C. A. Cell Size B. 900 fl 850 800 750 700 650 600 550 500 450 400 Cell number * 10 5 Cell Number 6 5 4 3 2 1 0 mm 1.2 1 0.8 0.6 0.4 0.2 0 Ammonia Production 4
Wellen_FigS4 A. B. % of glucose+ IL-3Ra Surface Expression 120 Glc- 100 Glc- GlcNAc+ 80 60 40 20 0 UT CSN DJ Tun Glucose + GlcNAc + + + pstat5 Stat5 ps6 S6 peif2a eif2a CSN Tun C. fl 900 800 700 600 500 400 Cell Size Ctrl CSN DJ Tun Glc- Glc-GlcNAc+ D. Arbitrary Units 140 120 100 80 60 40 20 0 IL-3Ra Surface expression *** Mgat5-wt Mgat5(L188R) 5
6 Wellen_FigS5
Wellen_FigS6 Ctrl BHA NAC GlcNAc + + + pstat5 (Tyr 694) Stat5 7
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 A) IL-3-dependent cells were withdrawn from glucose for 24 hours, then treated with either 15 mm glucose, 15 mm GlcNAc, or left untreated for an additional 24 hours.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 M-NSF-60 cells were withdrawn from glucose in the presence or absence of 15 mm GlcNAc for 2 days.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 Primary bone marrow cells were withdrawn from glucose in the presence or absence of 15 mm GlcNAc for 4 days and cell size was determined in fl (mean +/- s.d. of triplicate samples; ***, p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 IL-3-dependent bax-/-bak-/- cells were withdrawn from glucose for 3 days in the presence of 0.5 mm or 1 mm glucosamine or 15 mm GlcNAc.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
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
Table S1: Total metabolites (Labeled + Unlabeled) Hexosamine GlcNAc-P 1h 1242 2649 2997 3821 6182 4196 24h 2054 2799 4971 3494 3568 3742 72h 1078 2272 2831 2079 3864 4455 UDP-GlcNAc 1h 174655 195150 248424 250639 325033 295803 24h 164265 161710 83318 80807 355635 325021 72h 120929 126735 34527 40227 324055 374262 Glycolysis Lactate 1h 5549 6602 17561 23932 4166 6205 24h 4150 3120 57917 52843 5046 5169 72h 8170 5273 47742 45668 7213 8043 Hexose-P 1h 3045 1617 8395 8516 1138 1839 24h 784 893 2837 3731 1190 1189 72h 1183 972 1896 1950 833 1164 3-PG 1h 1924 1421 4287 3908 2052 1065 24h 424 590 448 815 889 213 72h 842 1212 1041 405 2053 746 FBP 1h 713 1071 12007 15986 1469 1045 24h 1162 449 2475 3063 583 1078 72h 591 619 3045 2224 1887 149 Pentose Phosphate Ribose-P 1h 473 549 3458 4031 681 551 24h 741 595 2917 2392 511 910 72h 277 214 699 896 172 354 PRPP 1h 500 1324 4713 13202 831 318 24h 465 140 11280 14510 0 0 72h 464 409 368 1005 845 2076 TCA Succinate 1h 6896 8106 12283 14220 9960 8762 24h 3608 3683 80714 67732 5789 4497 72h 4784 5354 32506 29507 13602 13129 Malate 1h 13988 17362 38437 46743 17462 22689 24h 4754 5364 44625 39816 9406 5319 72h 3294 3800 13572 11010 10408 8441 Ketoglutarate 1h 5403 5628 7198 6659 5372 5328 24h 3946 4555 8018 8305 5092 4513 72h 4065 3749 6994 7339 6633 5786 Citrate 1h 11880 12867 30318 25177 17391 15864 24h 1352 2194 12341 12123 3601 3025 72h 3985 6225 40808 30695 18619 17661 10
Other Glycerol-P 1h 7172 7680 10832 11287 7684 8033 24h 2510 2943 9441 9852 2575 3373 72h 2633 2912 2610 3502 2644 3151 Glutamate 1h 18916 19072 34978 24410 30027 29135 24h 22559 27006 78668 73553 28301 27081 72h 22773 17819 35458 33216 32034 28554 Reduced glutatione 1h 10514 11073 13167 13130 11723 17090 24h 39432 26829 31274 21515 28587 19558 72h 48754 56490 14007 13213 71674 54984 Oxidized glutatione 1h 3572 2409 3163 4148 2789 5217 24h 4169 2846 6152 5838 6470 2641 72h 5516 4591 2876 5483 8129 6134 N-acetyl-glutamate 1h 5876 2989 2750 4906 4026 2534 24h 3258 0 4171 4751 4850 0 72h 3267 4042 1771 1209 3967 3588 Ornithine 1h 1807 1615 3241 1035 1785 2186 24h 2204 1415 1869 1194 1800 1375 72h 1048 1961 5306 2682 983 2679 11
Table S2. Labeled metabolites Hexosamine no label 13 C-Glucose 13 C-GlcNAc GlcNAc-P 1h 0 0 139 408 1792 1691 24h 0 208 362 235 1759 1471 72h 0 0 235 345 2274 3280 UDP-GlcNAc 1h 0 0 74050 73699 103431 99013 24h 0 0 69362 67362 305754 285591 72h 0 0 29025 33055 302744 353889 Glycolysis Lactate 1h 0 0 12527 17557 0 0 24h 0 0 52586 46643 0 224 72h 0 0 42763 39312 280 0 Hexose-P 1h 461 541 6804 7290 0 389 24h 0 304 1819 1817 297 518 72h 154 364 1315 1369 0 526 3-PG 1h 0 0 2457 1630 0 0 24h 0 0 309 419 0 0 72h 0 0 452 191 358 201 FBP 1h 152 204 11745 15395 487 449 24h 0 0 1708 1818 137 0 72h 0 0 1040 1018 186 0 Pentose Phosphate Ribose-P 1h 0 0 2716 3448 0 0 24h 0 0 2383 1924 0 0 72h 0 0 506 742 0 0 PRPP 1h 0 0 4713 13202 0 0 24h 0 0 11280 14510 0 0 72h 0 0 368 1005 845 1686 TCA Malate 1h 1322 1640 12100 14100 1816 2123 24h 546 733 25780 23200 1293 551 72h 395 362 8656 7573 1294 836 Ketoglutarate 1h 0 107 713 162 0 0 24h 0 0 2257 2517 0 0 72h 141 0 1906 2260 109 0 Citrate 1h 1394 439 14068 12337 2081 3273 24h 0 0 10818 10294 161 184 72h 262 440 33722 27218 2319 2054 Other Glycerol-P 1h 0 0 2280 3432 0 0 24h 0 0 5300 5676 0 0 72h 0 0 978 1143 0 0 Glutamate 1h 0 0 8244 3795 595 0 24h 0 0 38277 37891 0 0 72h 0 0 17721 16467 337 537 12
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