Cell Reports, Volume 28

Supplemental Information

Glutamine Synthetase Promotes Radiation

Resistance via Facilitating Nucleotide

Metabolism and Subsequent DNA Damage Repair

Shujun Fu, Zhi Li, Lanbo Xiao, Wenfeng Hu, Lu Zhang, Bowen Xie, Qin Zhou, JunjuHe, Yanfang Qiu, Ming Wen, Yanni Peng, Jie Gao, Rong Tan, Yuezhen Deng, LiangWeng, and Lun-Quan Sun

Figure S1. Radiation resistant cancer cells reprogram cellular metabolic flux. Related to Figure 1.(A) Heat map of metabonomics of CNE2, CNE2-IRR, U251 and U251-IRR cell. The content for eachbiochemical factor in each sample is represented as increase in red or decrease in green (n≥4).(B) Pie charts indicating the percentage of biochemical factors with mean levels that were unchangedor significantly (p<0.05) increased or decreased.(C, E and F) Relative cellular levels of metabolites in U251 and U251-IRR cells (n=6).(D) Representative traces showing change in OCR and ECAR during mitochondrial and glycolysisstress test of U251, U251-IRR cells (n=4).Data are represented as mean ± SD. n represents experimental replicates. n.s p>0.05, *p<0.05,**p<0.01, ***p<0.001.

Figure S2. GS promoted cell survival after radiation. Related to Figure 2.(A) Heat map of transcriptomics of CNE2 and CNE2-IRR. The content for each gene expression ineach sample is represented as increase in red or decrease in blue (n=3).(B) Volcano Plot of transcriptomics of CNE2 and CNE2-IRR.(C) GO enrichment analysis of metabolic pathways, such as glucose, glutamine, lipid and nucleotidemetabolism.(D) GO enrichment analysis of pathways GS is involved in.(E) Apoptosis of U251-IRR cells with GS knockdown after 8Gy radiation.(F) Survival fraction of U251-IRR cells to 0, 2, 4, 6, 8, 10Gy radiation after GS was knocked down.Data are represented as mean ± SD. n represents experimental replicates. *p<0.05, **p<0.01.

Figure S3. GS facilitated nucleotide synthesis for DNA repair. Related to Figure 3.(A) Relative cellular nucleotide metabolites of CNE2, CNE2-IRR, U251 and U251-IRR cells (n≥5).(B) Relative cellular nucleotide metabolites of CNE2-IRR shcon, CNE2-IRR shGS1, U251-IRR shconand U251-IRR shGS1 cells (n≥5).(C) γH2A.X foci of CNE2-IRR shcon and shGS cells treated with normal (2mM) or high concentrationglutamine (4mM) at 6h after 8Gy. The scale bars represent 2.5μm.Data are represented as mean ± SD. n represents experimental replicates. n.s p>0.05, *p<0.05,**p<0.01, ***p<0.001.

Table S1 p-value, q-value and lFDR of genes from RNA sequencing. Related to Figure 2.Gene ID p-value q-value lFDRGS 0.0000974 0.0000804 0.000361399GLS 0.120863 0.03261319 0.3885743GLS2 0.02960996 0.009031987 0.09833308GLUD 0.001035973 0.000427536 0.003221275GOT1 0.4604877 0.1095139 1GOT2 0.002371866 0.000853353 0.00731315GPT 0.8636558 0.1954575 1GPT2 0.000149611 0.000110488 0.00052899ME1 0.000754532 0.00033085 0.002370909ME2 0.000176655 0.000118035 0.000614109

PDHA1 0.0000939 0.0000804 0.000350238PDHA2 0.4226497 0.1040421 1PDHB 0.0000757 0.0000758 0.000289972CS 0.0000341 0.0000478 0.000146514

ACO1 0.000974063 0.000414168 0.003033974ACO2 0.007158298 0.002511037 0.0226207IDH1 0.09958097 0.02794535 0.3247303IDH2 0.001081566 0.000433599 0.00335932OGDH 0.0000329 0.0000478 0.000142102SDHA 0.002028735 0.000749109 0.006252548SDHB 0.7925398 0.1823033 1SDHC 0.0000621 0.0000670 0.000244336SDHD 0.00127748 0.000497915 0.00395367FH 0.00030224 0.00016734 0.001002061

MDH1 0.000310077 0.00016734 0.001026021MDH2 0.000332384 0.000172735 0.001094116LDHA 0.07626314 0.02183845 0.2520762LDHB 0.01776436 0.005796748 0.05810962LDHC 0.02535063 0.007904593 0.08387124DLAT 0.01045822 0.003493912 0.03352262DLD 0.0000484 0.0000617 0.000197251CPS1 0.0000568 0.0000664 0.000226291OTC 0 0 0.000000259ASS1 0.000140862 0.000109806 0.00050126ARG1 0.01991197 0.006349871 0.06539365ARG2 0.0000231 0.0000462 0.000105564GSX1 0.4226497 0.1040421 1GSX2 0 0 0.000000259ACLY 0.0000268 0.0000469 0.000119505HMGCR 0.0000058 0.0000164 0.0000345

HMGCL 0.000202302 0.000129027 0.000694154HMGCS1 0.000299895 0.00016734 0.000994888HMGCS2 0.7456709 0.174381 1FASN 0.00189501 0.000718643 0.005840982

CACTIN 0.2928441 0.07470972 0.8301089HK1 0.000265097 0.000161726 0.000888197HK2 0.000474597 0.000214816 0.001525781HK3 0.1734304 0.04591478 0.5368859PFKM 0.06553587 0.0191576 0.2176795PKM 0.000399241 0.000200069 0.001297474G6PC3 0.000434231 0.000203097 0.001403575G6PC2 0 0 0.0000003G6PC3 0.000434231 0.000203097 0.001403575SCD 0.0000052 0.0000164 0.0000315PC 0.000164916 0.000115701 0.000577263

GYS1 0.0000151 0.0000352 0.0000743GYS2 0.2484181 0.06454947 0.7276451PYGM 0.976709 0.2175344 1PHGDH 0.0000954 0.0000804 0.000355048NOS1 0.445784 0.1078449 1NOS2 0.1099899 0.03026119 0.3562436NOS3 0.04245441 0.01267442 0.1416533SLC1A5 0.008779808 0.003004723 0.02795354

Table S2 Oligonucleoties. Related to Key Resourses Table.Oligonucleotides SOURCE IDENTIFIERGS-FW: TAAGGACCCTAACAAGCTGGT PrimerBank pga.mgh.harvard.

edu/primerbank/GS-RV: CCGTTTACAGGTGTGCCTCAA PrimerBank pga.mgh.harvard.

edu/primerbank/GLS-FW: TCTACAGGATTGCGAACGTCT PrimerBank pga.mgh.harvard.

edu/primerbank/GLS-RV: CTTTGTCTAGCATGACACCATCT PrimerBank pga.mgh.harvard.

edu/primerbank/GLS2-FW: TCTCTTCCGAAAGTGTGTGAGC PrimerBank pga.mgh.harvard.

edu/primerbank/GLS2-RV: CCGTGAACTCCTCAAAATCAGG PrimerBank pga.mgh.harvard.

edu/primerbank/GLUD-FW: CTGGCTTGGCATACACAATG PrimerBank pga.mgh.harvard.

edu/primerbank/GLUD-RV: GCTGTTCTCAGGTCCAATCC PrimerBank pga.mgh.harvard.

edu/primerbank/

STAT5A-FW: GCAGAGTCCGTGACAGAGGPrimerBank pga.mgh.harvard.

edu/primerbank/

STAT5A-RV: CCACAGGTAGGGACAGAGTCTPrimerBank pga.mgh.harvard.

edu/primerbank/

STAT5B-FW: GAACACCCGCAATGATTACAGTPrimerBank pga.mgh.harvard.

edu/primerbank/

STAT5B-RV: ACGGTCTGACCTCTTAATTCGTPrimerBank pga.mgh.harvard.

edu/primerbank/

EBF1-FW: TGGGGTTCGTGGAGAAGGAAPrimerBank pga.mgh.harvard.

edu/primerbank/

EBF1-RV: CACGTAGAAATCCTGCTCCGPrimerBank pga.mgh.harvard.

edu/primerbank/

PRDM1-FW:AAGCAACTGGATGCGCTATGTPrimerBank pga.mgh.harvard.

edu/primerbank/

PRDM1-RV:GGGATGGGCTTAATGGTGTAGAAPrimerBank pga.mgh.harvard.

edu/primerbank/

TFAP2C-FW: CTGTTGCTGCACGATCAGACAPrimerBank pga.mgh.harvard.

edu/primerbank/

TFAP2C-RV: CTCAGTGGGGTTCATTACGGCPrimerBank pga.mgh.harvard.

edu/primerbank/

Site1-FW: TGCAGAGTCGAGAGTGGGAPrimerBank pga.mgh.harvard.

edu/primerbank/

Site1-RV: CGAGCAGGCGGGCGGGTAAPrimerBank pga.mgh.harvard.

edu/primerbank/Site2-FW: CCGCTGGTGGCTTGGAC PrimerBank pga.mgh.harvard.

edu/primerbank/

Site2-RV: CTCCAGGGGCGGCGCTTPrimerBank pga.mgh.harvard.

edu/primerbank/shGS1-U:GATCCGGCACACCTGTAAACGGATATTCAAGAGATATCCGTTTACAGGTGTGCTTTTTTACGCGTG

GenomeRNAi www.genomernai.org/Index

shGS1-L:AATTCACGCGTAAAAAAGCACACCTGTAAACGGATATCTCTTGAATATCCGTTTACAGGTGTGCCG

GenomeRNAi www.genomernai.org/Index

ShGS2-U:GATCCGCCTTTGATCCTAAGCCCATTTCAAGAGAATGGGCTTAGGATCAAAGGTTTTTTACGCGTG

GenomeRNAi www.genomernai.org/Index

ShGS2-L:AATTCACGCGTAAAAAACCTTTGATCCTAAGCCCATTCTCTTGAAATGGGCTTAGGATCAAAGGCG

GenomeRNAi www.genomernai.org/Index

shGS1-L:AATTCACGCGTAAAAAAGCACACCTGTAAACGGATATCTCTTGAATATCCGTTTACAGGTGTGCCG

GenomeRNAi www.genomernai.org/Index

ShGS2-U:GATCCGCCTTTGATCCTAAGCCCATTTCAAGAGAATGGGCTTAGGATCAAAGGTTTTTTACGCGTG

GenomeRNAi www.genomernai.org/Index

ShGS2-L:AATTCACGCGTAAAAAACCTTTGATCCTAAGCCCATTCTCTTGAAATGGGCTTAGGATCAAAGGCG

GenomeRNAi www.genomernai.org/Index

shGS1-L:AATTCACGCGTAAAAAAGCACACCTGTAAACGGATATCTCTTGAATATCCGTTTACAGGTGTGCCG

GenomeRNAi www.genomernai.org/Index

ShGS2-U:GATCCGCCTTTGATCCTAAGCCCATTTCAAGAGAATGGGCTTAGGATCAAAGGTTTTTTACGCGTG

GenomeRNAi www.genomernai.org/Index