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1 DOI: 1.138/ncb3445 In the format provided by the authors and unedited. a b HIF1A Pearson R =.5419 P < H2AFX VEGFA d Pearson R = P < H2AFX Overexpression Vector -WT hr (1% O 2 ) VEGF GLUT1 MDA-231 (#) 1-99 patient samples Z score MCF1A shluc sh N H N H HIF1a VEGF GLUT1 mub-g g 2.5 j MDA shluc shrnf8 H2AFX N H N H =8 hr. f g MEF Cells RNF8 VEGF Mdc1 -/- Mdc1 +/+ MEF Cells hr. (1% O 2 ) ANG H2ax +/+ H2ax -/- 2 MDC1 GLUT hr. (1% O 2 ) GLUT1 GLUT4 GLUT1 GLUT4 ANG PKM2 ANG1 ANG2 l DAPI h MDA-231 shmdc1 shluc #1 #2 N H N H N H =8 hr. 2 MDC1 VEGF ANG1 PKM2 EPO i 9 GLUT H2AFX Pearson R =.49 P <.1 Pearson R =.6579 P <.1 MEF Cells Rnf8 +/+ Rnf8 -/ hr. (1 % O 2 ) RNF8 ANG GLUT1 c GLUT4 PKM2 Hypoxia Normoxia H2ax -/- H2ax +/+ H2ax -/- H2ax +/+ e k 2-NBDG Integration (a.u.) Lactate Production (mm/l) MDA-231: sh shluc #1 #2 #3 N H N H N H N H HIF1a HIF1a N H N H Glucose uptake shluc Normoxia Hypoxia sh Merged shluc sh Supplementary Figure 1 HIF1α signalling is positively correlated with expression but not with DNA damage markers. a, Heat map data were generated from analysis of biopsy specimens of 99 untreated patients with stage 1-3 breast cancer registered at MD Anderson Cancer Center (MDACC). Co-expression analysis was validated by using probes hybridized to, HIF1α, VEGF and PKM2 genes. b, The co-expression of HIF1α target genes was correlated with that of by the study of 5 untreated breast cancer patients at MDACC. c, Luc or knockdown MCF1A breast epithelial cells were cultured in normoxic or hypoxic conditions for 8 h, and lysates were collected for IB.d, Vector- or -overexpressing MDA-MB-231 cancer cells were cultured under hypoxia at the indicated times and harvested for IB. e, Luc or knockdown MDA-MB-231 cells were grown under normoxia or hypoxia for 24 h and treated with NBDG ( mm) for 2 h; glucose uptake was quantified by FACS analysis (n=3 independent experiments, upper). Luc or knockdown MDA-MB-231 cells were cultured under normoxia or hypoxia for 24 h and lactate production was measured (n=3 biological replicate samples, lower). (f-j) H2ax+/+ and H2ax-/- MEF (f) or Mdc1+/+ and Mdc1-/- MEF or LUC and MDC1 knockdown MDA-MB-231 (g, h) or Rnf8+/+ and Rnf8-/- MEF or LUC and RNF8 knockdown MDA-MB-231 (i, j) cells were cultured in hypoxic conditions at the indicated times and harvested for IB of hypoxia-inducible protein expression. k, Luc or knockdown MDA-MB-231 cells silenced by three different shrnas were cultured in normoxic (N) or hypoxic (H=8 hours) conditions and harvested for detection of HIF1α expression level. l, H2ax+/+ and H2ax-/- MEF cells were cultured in hypoxic or normoxic conditions for 12h, and the localization of HIF1α and proteins was visualized by immunofluorescence microscopy. Image represents 1 out of 3 independent experiments.. Scale bar= µm. Statistical significance of triplicated results was assessed by two tailed t-test. Mean ± SD. P<.1 was considered significant. Unprocessed original scans of blots are shown in Supplementary Fig Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

2 a c DAPI MRE11 g Merged Overexpression + Mock SHARP1 SHARP1 N H N H N H N H HIF1a HA-SHARP1 HA- MDA-231 Hypoxia Normoxia H2ax -/- H2ax +/+ H2ax -/- H2ax +/+ d DAPI RNF8 g Merged b Overexpression HA-SHARP1: HA-: Flag-HIF1a: T Flag-HIF1a HA-SHARP1 mub- HA- Hypoxia Normoxia H2ax -/- H2ax +/+ H2ax +/+ H2ax -/- H2ax +/+ 1X 1X 1X 1X 1X 1X 1X 1X Supplementary Figure 2 SHARP1 may exhibit a competition with. a, Mock or or SHARP1 or along with SHARP1 were transiently overexpressed in MDA-MB-231 cells were cultured under hypoxic conditions for 8 hours and harvested for IB analysis. b, Lysates of 293T cells transfected with (Flag)-HIF1α along with HA- or (HA)-SHARP1 were subjected to IB analysis. c, d, H2ax+/+ and H2ax-/- MEF cells were cultured in normoxic or hypoxic conditions for 12h, and the localization of MRE11 (c) or RNF8 (d) and γ proteins was visualized by immunofluorescence microscopy. Scale bar= µm. γ is induced upon hypoxia without colocalization with DNA damage factors such as MRE11 and RNF8. The experiments a-d performed once. Unprocessed original scans of blots are shown in Supplementary Fig Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

3 a E3 ligase WCE - - Ni-NTA IB: HAmUb- HA- Smurf1/ Smurf2 c-iap2 c-iap1 FBW7 Itch MDM2 SKP2 His-Ub HA b e Stable overexpression, MDA-231 pmx -WT mub- DAPI HIF1a g Merged c HA- - WCE Ni-NTA His-Ub d IB: HAmUb- HA- IP: IgG N H Input N H MDA-231 HIF1a g HIF1a g Hypoxia Normoxia H2ax -/- H2ax +/+ H2ax -/- H2ax +/+ f Human Bovine Mouse Salmon Zebrafish Arabidopsis Drosophila Human Bovine Mouse Salmon Zebrafish Arabidopsis Drosophila Human Bovine Mouse Salmon Zebrafish Arabidopsis Drosophila MSGRGKTGGKARAKAKSRSSRAGLQFPVGRVHRLLRKGHYAE-RVGAGAPVYL MSGRGKTGGKARAKAKSRSSRAGLQFPVGRVHRLLRKGHYAE-RVGAGAPVYL MSGRGKTGGKARAKAKSRSSRAGLQFPVGRVHRLLRKGHYAE-RVGAGAPVYL MSGRGKTGGKARAKAKSRSSRAGLQFPVGRVHRLLRKGNYAH-RVGAGAPVYM MSGRGKAVSKTRAKAKTRSSRAGLQFPVGRVHRLLRKGNYAH-RVGAGAPVYL MSTGAGSGTTKGGRGKPKATKSVSRSSKAGLQFPVGRIARFLKSGKYAE-RVGAGAPVYL -----MAGGKAGKDSGKAKAKAVSRSARAGLQFPVGRIHRHLKSRTTSHGRVGATAAVYS :: :::: :. :.. K74/ AAVLEYLTAEILELAGNAARDNKKTRIIPRHLQLAIRNDEELNKLLGGVTIAQGGVLPNI AAVLEYLTAEILELAGNAARDNKKTRIIPRHLQLAIRNDEELNKLLGGVTIAQGGVLPNI AAVLEYLTAEILELAGNAARDNKKTRIIPRHLQLAIRNDEELNKLLGGVTIAQGGVLPNI AAVLEYLTAEILELAGNAARDNKKSRIIPRHLQLAVRNDEELNKLLGGVTIAQGGVLPNI AAVLEYLTAEILELAGNAARDNKKSRIIPRHLQLAVRNDEELNKLLGGVTIAQGGVLPNI SAVLEYLAAEVLELAGNAARDNKKTRIVPRHIQLAVRNDEELSKLLGSVTIANGGVLPNI AAILEYLTAEVLELAGNASKDLKVKRITPRHLQLAIRGDEELDSLIK-ATIAGGGVIPHI ::::::. ::...:. :: K119/1 K134/135 S139 QAVLLPKKTSATVGPKAPSGGKKATQASQEY QAVLLPKKTSATVGPKAPAGGKKATQASQEY QAVLLPKKSSATVGPKAPAVGKKASQASQEY QAVLLPKKTGAAAAPSGKA-GKKASSQSQEY QAVLLPKKSSGGVSTSG----KKSSQQSQEY HQTLLPSK VGKNKGDIGSASQEF HKSLIGKK----EETVQDPQRKGNVILSQAY : :..:.. Supplementary Figure 3 is an E3 ligase which monoubiquitinates and interacts with HIF1α. a, Lysates of 293T cells transfected with hemagglutinin-tagged (HA-) and His-Ub, along with various E3 ligases, were subjected to in vivo ubiquitination assay and IB analysis. b, Vector (pmx)- or -overexpressing MDA-MB-231 cells were cultured in normoxic or indicated hypoxic time points and subjected to lysis followed by IB analysis. c, 293T cells co-transfected with His-Ub, and HA- or various mutants were subjected to lysis for in vivo monoubiquitination assay. ubiquitination sites were found at Lys (K) 119 and K1. d, Lysates of MDA-231 cells cultured in normoxic or hypoxic conditions for 4 h were immunoprecipitated with control or antibody followed by IB. e, Immunofluorescence assay of the localization of HIF1α and γ proteins from H2ax+/+ and H2ax-/- MEF cells cultured in hypoxic or normoxic conditions for 12 h. Scale bar= µm. Image represents 1 out of 3 independent experiments. f, Multiple alignments of amino acids are shown; ubiquitination sites K119/1 (highlighted in red letters) and the phosphorylation site S139 (in the SQEY motif, highlighted in blue) are highly conserved among different species. Numbers show the positions of K (lysine) and/or S (serine). Unprocessed original scans of blots are shown in Supplementary Fig Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

4 a MDA-231 shluc shhif1a hr. (1%O 2 ) HIF1a VEGF GLUT1 GLUT4 g b mrna Expression Fold MEF/Traf6 2 4 c Labeled DNA + Labeled Lysate + Labeled DNA + Cold DNA Lysate DNA Biotin labeled d Traf6 6 bp ChIP: HIF1a Input IgG N H N H (-) Hela cells IP:GAPDH Input 3 e f EPO mrna Pearson R =.3396 P < mrna ANGPT1 mrna Pearson R =.3517 P < mrna Count Count MDA-231-shLUC 1 2 PE-A (x 1,) MDA-231-sh#1 ANGPT2 mrna Pearson R =.3628 P < mrna SLC2A1 mrna Pearson R =.4564 P < mrna g Count PE-A (x 1,) MDA-231-sh#2 1 2 PE-A (x 1,) SLC2A4 mrna Pearson R =.4327 P < mrna PDK1 mrna Pearson R =.21 P < mrna Count (%) 6 4 Cell Cycle Profile ns ns G/G1 S G2/M Poly shluc sh#1 sh#2 Supplementary Figure 4, a hypoxia responsive gene, regulates HIF1α signalling. a, MDA-MB-231 cancer cells in which Luc or HIF1α was silenced by shrna were cultured in normoxic or hypoxic conditions for 8 h, and lysates were collected for IB analysis. b, Primary MEF cells cultured in hypoxic conditions for 2 and 4 h were subjected to qpcr of Traf6 mrna (n=3, biologically independent extracts).. c, Lysates of HEK-293 cells were treated with biotin-labeled -DNA or along with biotin-labeled - DNA and cold DNA (not Labeled) compared to labeled-dna alone were subjected to electrophoretic mobility shift assay (EMSA). d, Hela cells were cultured in normoxic or hypoxic conditions and harvested for chromatin immunoprecipitation (ChIP) with HIF1α antibody followed by qpcr for the expression of genes and were compared to GAPDH control. e, Scatter plots were generated for the correlated expression of HIF1α target genes with. Tumour tissues were used from 5 untreated patients with stage 1-3 breast cancer. The biopsies were obtained through fine-needle aspiration. Expression analysis was carried out by Nexus Expression.3 software (Biodiscovery). f, g, LUC or knockdown MDA-MB-231 cells were subjected to cell cycle analysis (n=3, independent experiments). Statistical significance of three biological replications was assessed by two tailed t test. Mean ± SD, p<.1 was considered significant. Unprocessed original scans of blots are shown in Supplementary Fig Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

5 a MEF/H2ax -/- /Rescued b vector WT KR SA hr. (1% O 2 ) VEGF GLUT1 g shluc sh#1 sh#2 --- MG MG MG132 N H N H N H N H N H N H (=4 hr.) HIF1a MDA-231 c -SA -KR -WT Vector DAPI HIF1a Merged d 2 2 MEF N H N KU H (=12 hr.) patm (S1981) ATM g HIF1a e 2 2 MEF ATM-WT ATM-Null hr. (1% O 2 ) patm ATM HIF1a ANG VEGF PKM2 f 2 2 IP: ATM NM M N H N H N H In put mub- ATM ATM g g MEF/Traf6 -/- /Rescued Vector WT CA 1% O2: hr. g Chromatin WCE a-tubulin h Expression Value Vector WT KR SA ChIP: anti-hif1a for VEGF Expression N H N H N H N H 293T Supplementary Figure 5 mub- and γ formation are critical for HIF1α signalling. a, H2ax / MEF cells in which was restored with vector, -WT or -KR mutant construct were cultured under hypoxic conditions for the indicated time points and subjected to lysis and IB. b, -silenced MDA-MB-231 cells were treated with vehicle or MG132 in hypoxic conditions for 4 hours and subjected to lysis and IB. c, H2ax-/- MEF cells reconstituted with vector, -WT or -KR or -SA mutant were incubated under hypoxic conditions for 12 h, and the localization of HIF1α was visualized by confocal microscopy. Scale bar= μm. Experiments were performed twice. d, MEF cells cultured in normoxic and hypoxic conditions were treated with or without ATM inhibitor KU55933 for the indicated times, harvested and subjected to lysis for IB. e, MEF cells generated from Atm+/+ or Atm-/- mice cultured in hypoxic conditions for 8 h and subjected to lysis and then to IB analysis. f, 293T cells treated with non-modified (NM) and modified (M, ub-conjugated) peptides (2 ng/ml) were cultured in normoxic or hypoxic conditions for 4 h, and lysates were collected for IP with ATM antibody, followed by IB. Interaction of ATM to mub- was neutralized followed by reduction in γ expression. g, Traf6 / MEF cells restored with vector, -WT or -C7A mutant were cultured in hypoxic conditions for various time points and harvested for chromatin fractionation and IB. γ formation was shown in the chromatin fraction of the cells rescued with -WT. h, 293T cells stably overexpressing vector, -WT or -KR or -SA mutant were cultured in normoxic or hypoxic conditions and harvested for ChIP-qPCR of VEGF using HIF1α antibody (n=3, biologically independent extracts). Results are representative from three biological replications. Statistical significance was assessed by ANOVA. Unprocessed original scans of blots are shown in Supplementary Fig Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

6 a b hr Cell Number (X) SA -KR -WT Vector NS Day Vector -WT -KR -SA c pbabe HIF1a-WT HIF1a-Mut sh shluc d e 6 f 4 VEGF Concentration (x1 pg/ml) Normoxia Hypoxia shluc sh#1 sh#2 N H N H N H % Migration 4 shluc sh shluc sh#1 sh#2 % Invasion 3 shluc sh shluc sh#1 sh#2 g 1 5 Nonubiquitinated peptide 1 5 Ubiquitinated peptide Comp-PE-A Comp-PE-A Comp-FITC-A Comp-FITC-A Supplementary Figure 6 -targeting reduces cancer cell survival, migration and tumourigenesis. a, MDA-MB-231 cells stably overexpressing vector, -WT or -KR or -SA mutant were harvested for cell viability assay (n=3, biological replicate samples). b, knockdown MDA-MB-231 cells rescued with -WT or an mutant were seeded in triplicate for a wound healing analysis using confocal microscopy. c, Luciferase expressing MDA-MB-231 cancer cells in which was silenced by shrna and then restored with vector, HIF1α-WT or HIF1α- Mut were injected into the mammary fat pad of nude mice and luciferase expression in tumour growth was counted. d, Luc or knockdown MDA-MB-231 breast cancer cells were cultured in normoxic or hypoxic conditions for 24 h, and the conditioned media were collected for detection of secreted VEGF by ELISA assay (n=4, biological replicate samples). e-f, MDA-MB-231 cells in which Luc or was silenced with two different shrnas and plated in transwell chambers for the cell migration (e) and invasion (f) assay (n=4, biological replicate samples). Scale bar= µm. g, MDA-MB-231 cells were treated with non-ubiquitinated (left, CVLLPKKTSAT) and ubiquiitnated (right, CVLLPK(ub-K) TSAT) peptides (1 ng/ml) overnight, and cell viability was determined by flow cytometry. Statistical significance was measured using two tailed t-test (e-f) and ANOVA (a, d). Mean ± SD, P<.1 and P<.5 were considered significant Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

7 a Biological processes Cancer hallmarks Activation of MAPKKK pathway Cell migration Pyridine biosynthesis Suppression of cell cycle checkpoint Wnt signaling pathway Suppression of apoptosis by intracellular signals Glucose metabolism Glucose transport Cell proliferation Growth hormone secretion Suppression of caspase activation via cytochrome c EGFR signaling pathway Positive regulation of I-κB/NF-κB cascade Telomere maintenance Fatty acid biosynthesis Angiogenesis Suppression of apoptotic program STOP% " " Sustaining proliferation Resisting cell death Tissue invasion and metastasis Replicative immortality Angiogenesis Continuous growth signaling Metabolic reprogramming Evading immune surveillance Cancer-promoting inflammation Genome instability b Sustaining proliferation JNK cascade Positive regulation of JNK cascade I-kB/NF-kB signaling cascade Cell proliferation Activation of MAPKKK activity Activation of NF-kappaB-inducing kinase G2 phase of mitotic cell cycle DNA-dependent DNA replication Activation of MAPK activity Activation of MAPKK activity Activation of JNK activity Positive regulation of I-kB/NF-kB signaling cascade Positive regulation of cell proliferation EGFR pathway S phase of mitotic cell cycle Enrichment score c Metabolic reprogramming Lipid biosynthetic process Fatty acid biosynthetic process Fatty acid metabolic process Amino acid biosynthetic process Lipid metabolic process Glutamate transport Purine ribonucleoside monophosphate biosynthetic process Glucose transport Glucose metabolic process GTP biosynthetic process Pyridine nucleotide biosynthetic process Glutamate signaling pathway Lipopolysaccharide biosynthetic process Coenzyme A biosynthetic process Enrichment score d Tissue invasion and metastasis Regulation of cell adhesion Endothelial cell migration Cell migration Wnt receptor signaling pathway Enrichment score Supplementary Figure 7 Overexpression of is associated with upregulation of major cancer hallmarks. a, Transcriptomics gene expression profiles of 2 breast cancer patients (cohort GSE194, Gene Expression Omnibus database, were analyzed using the Nexus Expression 3. software (BioDiscovery). The gene expression profiles of the highest quartile were compared with the lowest quartile, and then matched with related biological processes and corresponding cancer hallmarks. The size of cancer hallmark symbols signified the magnitude of their upregulation when expression was elevated in breast cancer. b-d, The bar graph indicates a significant increase in cancer proliferation (b), metabolomics reprogramming (c), tissue invasion and metastasis (d) which associated with enhanced expression. Enrichment scores were calculated using the Nexus Expression 3. software (BioDiscovery). This Circos map was built using the Circos software ( Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

8 a shluc/vector sh/vector sh/hif1a-wt sh/hif1a-mut 1 Count b Tumour Volume (x mm 3 ) shluc/pbabe sh/pbabe sh/hif WT sh/hif Mut W3 W4 W5 ns c shluc sh/hif1a Vec Vec WT Mut Tumour weight (g) ns MSCV- MSCV- d vector mir-145 e Tumour Volume (x mm 3 ) W1 MSCV mir-145 ns W2 W3 W4 W5 W6 f Supplementary Figure 8. regulates HIF1α-dependent breast cancer development. a-c, knockdown MDA-MB-231 cells (expressing luciferase) rescued with pbabe vector, HIF1α-WT or HIF1α-Mut (constitutively active mutant) were injected into the mammary fat pad of nude mice (n=5/group) and tumour sizes were monitored weekly (b) and tumour weight was measured at week 6 (c, n=5 tumour/group, Mean ± SD) d, MSCV-vector or MSCV-miR-145 overexpressing MDA-MB-231 breast cancer cells were subcutaneously injected into the right flank of 6-weekold nude mice (n=4/group) and tumour volume was measured weekly. e, Primary tissues, cell lines in different human disease with different types of mutation which is involved in functional effect, inheritance mode, translation impact, unclassified mutation, zygosity and wild type were analysed using Ingenuity Pathway ( The publication data range was from Jan Apr. 16. f, Activation of, and HIF1α network in cancer genomics was proven by cbioportal web-based data sets ( Panels e and f indicate that this network is significantly existed including, and HIF1α gene pairs with co-occurring alterations in many cancer types. Statistical significance was assessed by ANOVA. Mean ± SD. ns, not significant. P<.1 was considered significant Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

9 Figure 2b Figure 2c 1 1 Figure 2f Supplementary Figure 9 Unprocessed scans of full blots. The unprocessed films showing the key experiments displayed in main figures and Supplementary figure Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

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22 Supplementary Table Legends Supplementary Table 1 Associations of,, γ, HIF1α, VEGF and GLUT1 expression with tumour status. In 212 breast carcinomas, high expression levels of,, γ, HIF1α, VEGF and GLUT1 were significantly associated with increments of primary tumour status (pt, p.2), nodal metastasis (pn, p.2) and stage (p.6). Supplementary Table 2 Associations between,, γ, HIF1α, VEGF and GLUT1 expression. expression was significantly positively correlated with expression of, γ, HIF1α, VEGF and GLUT1. Interestingly, the labelling indexes of all six of these markers are significantly and positively correlated to each other except for HIF1α and GLUT1, whose correlation is not significant. Supplementary Table 3,, γ, HIF1α, VEGF and GLUT1 expression significantly associated with patient survival outcome. Together with pt status, pn status, and stage,,, γ, HIF1α and VEGF expression significantly predicted disease-specific survival (DSS) and metastasis-free survival (MeFS) by Cox proportional hazards model. GLUT1 expression was significantly predictive for MeFS but not DSS. By log-rank analysis,,, γ and VEGF expression were significantly predictive for both DSS and MeFS, while HIF1α and GLUT1 were predictive for poorer MeFS but not DSS. Supplementary Table 4 Multivariate survival analyses. High expression of and and higher tumour stage remained prognostically significant for DSS (, p=.17;, p<.1) and MeFS (, p=.42;, p=.45). Supplementary Table 5 shrnas, primers and probes used in experimental assays. a, shrnas used for silencing. b, Primers used for real time-quantitative PCR. c, Primers and probes used for ChIP and EMSA assays. Supplementary Table 6 Primary antibody list. Supplementary Table 7 Statistics source data. Supplementary References 1. Gao, J., et al. Integrative analysis of complex cancer genomics and clinical profiles using the cbioportal. Sci Signal. 6, 1-19 (13). 2. Cerami, E., et al., The cbio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data. Cancer Discov. 2, (12) Macmillan Publishers Limited, part of Springer Nature. All rights reserved.