Mouse primary acinar cells were isolated and cultured as described in). Briefly, all procedures for isolating acinar cells were performed under sterile conditions. Pancreases from 8-week-old male mice were mechanically and enzymatically digested with collagenase IA solution (1x HBSS containing 10 mM Hepes, 200 units/ml of collagenase IA, and 0.25 mg/ml of trypsin inhibitor) to obtain isolated acinar structures. Acini were grown in a medium containing one volume of glucose-free DMEM and one volume of F12 medium supplemented with 2.5 % FBS, 1% penicillin/streptomycin mixture, 0.25 mg/ml of trypsin inhibitor, and 25 ng/ml of recombinant human epidermal growth factor (EGF). After culturing on type I collagen-coated six-well culture dishes for 6 days, acinar to ductal-like cells were trypsinized and replated into another type I collagen-coated plate for subsequent experiments.

To establish the cell line that stably expresses the vector control, RRM1-WT, or RRM1-T734A, HPDE and HPNE cells were infected with the lentivirus containing the indicated cDNA and were selected with 2 μg/ml blasticidin. To establish the cell line that stably expresses LacZ shRNA or OGT shRNA , HPDE and HPNE cells were infected with the lentivirus containing the indicated shRNA and were selected with 1 μg/ml puromycin. To establish the cell line that stably expresses LacZ shRNA or PFKL shRNA , MCF-10A cells were infected with the lentivirus containing the indicated shRNA and were selected with 2 μg/ml puromycin. To establish the cell line that stably expresses LacZ shRNA or GFPT1 shRA , HPDE cells were infected with the lentivirus containing the indicated shRNA and were selected with 1 μg/ml puromycin. To establish the cell line that stably expresses LacZ shRNA , GLUT1 shRNA , GLUT2 shRNA , SGLT1 shRNA , or SGLT2 shRNA , HPNE cells were infected with the lentivirus containing the indicated shRNA and were selected with 1 μg/ml puromycin. To establish the cell line that stably expresses the vector control or GLUT1, MCF10A cells were infected with the lentivirus containing the indicated cDNA and were selected with 2 μg/ml blasticidin.

The non-transformed foreskin fibroblast cells (HS68) generated from newborn male foreskin tissue were grown in low glucose (5.5 mM) DMEM medium supplemented with 10% FBS and antibiotics (penicillin/streptomycin). All cell lines were regularly checked for mycoplasma infection. For regular glucose treatment, cell medium was refreshed every 36 hours, and cells were replated every 72 hours to avoid over-crowding. For high-glucose treatment, ribonucleosides (N) addition and deoxynucleosides (dN) addition, cell medium was refreshed every 36 hours.

The non-transformed mammary epithelial cells (H184B5F5/M10) generated from a 21-years old female mammary gland and non-transformed colon epithelial cells (NCM356) generated from a 65-years old male colon tissue were grown in MEM medium and M3™ base medium (INCELL) supplemented with 10% FBS and antibiotics (penicillin/streptomycin), respectively.

All cells were maintained at 37°C in a humidified atmosphere containing 5% CO 2 . The non-transformed human pancreatic ductal epithelial cells (HPDE) generated from a 75-years old male pancreatic specimen (a gift from Dr. Kelvin K. Tsai, National Health Research Institutes, Taiwan) were grown in keratinocyte serum-free (KSF) medium with 0.2 ng/ml EGF and 30 μg/ml bovine pituitary extract (Invitrogen Life Technologies). The non-transformed human pancreatic acinar-to-ductal epithelial-like cells (HPNE) generated from a 52-years old male pancreatic specimen and non-transformed lung epithelial cells (NL20) generated from a 20-years old female lung specimen were obtained from Dr. Michael Hsiao at the Genomics Research Center, Academia Sinica, Taiwan. HPNE cells were grown in a medium containing one volume of M3™ base medium (INCELL) and three volumes of glucose-free DMEM supplemented with 5% FBS, 5.5 mM glucose, 10 ng/ml EGF, and antibiotics (penicillin/streptomycin). NL-20 cells were grown in a medium containing one volume of glucose-free DMEM and one volume of F12 medium supplemented with 0.1 mM nonessential amino acids, 0.005 mg/ml insulin, 10 ng/ml EGF, 0.001 mg/ml transferrin, 500 ng/ml hydrocortisone, 4% fetal bovine serum and antibiotics (penicillin/streptomycin). The non-transformed human mammary epithelial cells (MCF-10A) generated from a 36-years old female mammary gland were grown in a medium containing one volume of glucose-free DMEM and one volume of F12 medium supplemented with 5% (vol/vol) donor horse serum, 20 ng/mL epidermal growth factor, 10 μg/mL insulin, 0.5 μg/mL hydrocortisone, 100 ng/mL cholera toxin, and antibiotics (penicillin/streptomycin).

We chose our sample sizes based on those commonly used in this field without predetermination by statistical methods. This is stated in the figure legends.

All experiments using animals were approved by the Institutional Animal Care and Utilization Committee of Academia Sinica, Taipei, Taiwan (IACUC#10-04-065). Male C57BL/6JNarl mice at the age of 4 weeks were obtained from the National Laboratory Animal Center (Taiwan). Unless specified otherwise, mice were maintained in a SPF (specific pathogen-free) animal facility at 20±2°C with a 12/12 hr light/dark cycle and had free access to water and standard laboratory chow diet. For establishing the hyperglycemia model, mice (n=72) with similar body weight and blood glucose levels were randomly allocated to two groups: one group was fed a chow diet (13% calorie from fat, LabDiet 5010) and another was fed a high-sucrose (17.5%) and high-fat (35.8%) diet (HS/HF diet, TestDiet 58R3). A blood glucose meter (Roche Life Science) was used to monitor the level of blood glucose every week. Twelve mice from each group were sacrificed at 5, 10, and 20 weeks after feeding. Pancreatic, small intestinal, colon, liver, lung, and kidney tissue samples were collected, fixed in 10% paraformaldehyde, embedded in paraffin, and cut into 5 μm sections for immunohistochemistry analysis.

All male PDAC patients, in age from 43 to 79 years old, had anatomically resectable PDAC (TNM stages I and II). In addition, all patients with DM had at least 5 years with DM prior to PDAC diagnosis. The non-tumor regions of pancreatic and intestinal tissues used for the study were judged by cell morphology with Hematoxylin and Eosin (H&E) staining by an experienced clinical pathologist.

All pancreatic cancer tissue specimens were from the National Taiwan University Hospital, Taipei, Taiwan. All patients were given informed consent, which was approved by the Institutional Review Board of the NTUH (201303029RINC) and NTUH (201411085RINB).

Methods Details

Plasmids, Construction of Expression Plasmids, and Site-Directed Mutagenesis The lentiviral shRNA expression vectors of pLKO.1-shLacZ, shOGT (TRCN35064, 35067), shPFK (TRCN342355), and shGFAT1 (TRCN75219, TRCN 75220) were from the National RNAi Core Facility (Taipei, Taiwan). pET28a-RRM1 and pET28a-RRM2 were kindly provided by Dr. Yun Yen at Taipei Medical University, Taipei, Taiwan. pCMV2-RRM1, pAS3w-mCherry-RRM2, and pAS3w-mCherry-RRM2B was kindly provided by Dr. Zee-Fen Chang at National Taiwan University, Taipei, Taiwan. E. coli BL21 Tuner (DE3) GST-OGT-WT and H568A competent cells were kindly provided by Dr. Hsiu-Ming Shih at the Institute of Biomedical Sciences in Academia Sinica, Taipei, Taiwan. The retroviral cDNA expression vectors of pWZL-Neo-Myr-Flag-PFKL and PFKM were from Addgene. pET28a-RRM2B was constructed by insertion of cDNA of RRM2B at BamHI site and EcoRI site of pET28a vector. The pCMV2-RRM1 (S253A, S616A, T734A, and S764A) mutant was generated using a QuickChange XL site-directed mutagenesis kit (Stratagene). pAS5w-RRM1-WT, pAS5w-RRM1-T734A, and pAS5w-GLUT1 were constructed by insertion of cDNA of RRM1 and GLUT1 at NheI and EcoRV sites of the pAS5w.bsd vector, respectively.

Immunohistochemistry (IHC) IHC was performed on a BenchMark Ultra auto-stainer (Ventana Medical Systems). Sections were deparaffinized with EZPrep buffer (Ventana Medical Systems), and antigen retrieval was also performed in this instrument. Slides were incubated with anti-γH2AX antibody (1:100, Cell Signaling #9718) or anti-O-linked N-Acetylglucosamine antibody [CTD 110.6] (1:100, Cell Signaling #9875) for 4 hours followed by 1-hour incubation with secondary antibody. Then slides were processed with a DAB detection kit (Ventana Medical Systems) and counterstained with haematoxylin (Ventana Medical Systems). For IHC data analysis, slides were scanned at 40× magnification using Aperio Digital Pathology Slide Scanners and the images of high power field at 40× magnification were randomly selected and analyzed by Leica Aperio Imagescope digital slide viewer v9.1.19.1568. Quantification of γH2AX and O-GlcNAc per sample was determined by analyzing 50 randomly chosen high-power fields (HPF), 40x magnification, and performed by Aperio IHC Nuclear Algorithm and Aperio Positive Pixel Count, respectively.

Immunofluorescence Staining For γH2AX and 53BP1 foci staining, cells grown on coverslips or culture dishes were fixed with 4% paraformaldehyde and permeabilized with 0.3% Triton X-100 in TBS (50 mM Tris-HCl, pH 7.4, 150 mM NaCl) buffer. For KRASG12D staining, cells grown on coverslips were washed with CSK (100 mM NaCl, 300 mM sucrose, 10 mM PIPES pH 7.0, 3 mM MgCl 2 , 0.1% of Triton X-100) buffer twice and then fixed with 4% paraformaldehyde, permeabilized with 0.3% Triton X-100 in TBS buffer. Cells were blocked with MAXblock™ (Active Motif) for 1 hour at 37°C, followed by staining with anti-γH2AX (1:1000, Merck Millipore #05-636), anti-53BP1 (1:1,000, Merck Millipore #MAB3802), or anti-KRASG12D (100 ng/μl, 1:100, Merck Millipore # PC10L) at 4°C overnight. After extensive washing, these cells were stained with Alexa Fluor 488-conjugated secondary antibodies for 1 hour at room temperature. After wash, cells were stained with DAPI (10 μg/ml, 1:1000, Life Technologies) and mounted with ProLong Gold Antifade Mounting Oil. The percentage of γH2AX and 53BP1 foci-positive cells was determined by analyzing 2,000 randomly chosen cells with Cellomics ArrayScan VTI and the foci-positive cells were defined as nuclei with at least one large focus or a granular pattern of green fluorescence.

Soft Agar Colony Formation Assay Soft agar colony formation assay was performed by seeding 104 cells in a layer of 0.35% agar/complete growth medium over a layer of 0.5% agar/complete growth medium in the wells of a 12-well plate. Cell medium containing the indicated concentration of glucose, nucleosides, deoxynucleosides, or Alloxan (Sigma-Aldrich) was replenished every 3 days. Cultures were maintained in a humidified 37°C incubator. On day 21 or day 27 after seeding, cells were fixed with pure ethanol containing 0.05% Crystal Violet (crystal violet (Sigma-Aldrich C3886), and the colony-forming efficiency was quantified under a light microscopy. Colonies, which size was quantified by the diameter of each colony, larger than 50 μm were counted and analyzed.

Glucose Metabolite Analysis Su et al., 2014 Su M.A.

Huang Y.T.

Chen I.T.

Lee D.Y.

Hsieh Y.C.

Li C.Y.

Ng T.H.

Liang S.Y.

Lin S.Y.

Huang S.W.

et al. An invertebrate Warburg effect: a shrimp virus achieves successful replication by altering the host metabolome via the PI3K-Akt-mTOR pathway. Following culture with the indicated concentrations of glucose for 3, 9, or 27 days, cells were washed twice with PBS and harvested with nuclease-free water. The samples were collected, sonicated with amplitude 80% around 30 sec (Hielscher, Ultrasound Technology), and then centrifuged at 10,000 g for 10 min. 100% acetone nitrate was added to the supernatant at a ratio of 1∶3. After being centrifuged again at 10,000 g for 10 min, the supernatant was lyophilized and the pellet was dissolved in 35 μl ddH2O for LC-ESI/MS metabolomic analysis described as follows by Su et al. (). To enhance the detection of the carboxylic acid and organic phosphate signals, 5 μl aniline/HCl reaction buffer (0.3 M aniline [Sigma-Aldrich, USA] in 60 mM HCl) and 5 μl of 20 mg/ml N - (3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC; Sigma-Aldrich, USA) were added to each sample of the hemocyte residue. Each mixture was vortexed and incubated at 25°C for 2 h, after which the reaction was stopped by adding 5 μl of 10% ammonium hydroxide. The aniline derivatized samples were then analyzed using an LC-ESI-MS system consisting of an ultra-performance liquid chromatography (UPLC) system (Ultimate3000 RSLC, Dionex) and a quadrupole time-of-flight (TOF) mass spectrometer with an the electrospray ionization (ESI) source (maXis UHR-QToF system, Bruker Daltonics). The cells metabolites were separated by reversed-phase liquid chromatography (RPLC) on a BEH C18 column (2.1×100 mm, Walters). The LC parameters were as follows: autosampler temperature, 4°C; injection volume, 10 μl; and flow rate, 0.4 ml/min. After pre-starting with 1% mobile phase B (0.1% formic acid in ACN) for 4 min, the elution started from 99% mobile phase A (0.1% formic acid in ddH2O) and 1% mobile phase B (0.1% formic acid in ACN). After holding at 1% for 0.5 min and raising to 60% over 5 min, mobile phase B was further raised to 90% in another 0.5 min, held at 90% for 1.5 min, and then lowered back to 1% in 0.5 min. The column was then equilibrated by pumping 99% B for 4 min. The acquisition parameters for LC-ESI-MS chromatograms were as follows: dry gas temperature, 190°C; dry gas flow rate, 8 L/min; nebulizer gas, 1.4 bar and capillary voltage, 3,500 V. Mass spectra were recorded from m/z 100–1000 in the negative ion mode. Data were acquired by HyStar and micrOTOF control software (Bruker Daltonics) and processed by DataAnalysis and TargetAnalysis software (Bruker Daltonics). Each metabolite was identified by matching with its theoretical m/z value and with the isotope pattern derived from its chemical formula. The identified metabolites were quantified by summing the corresponding area of the extracted ion chromatogram.

Cellular dNTP Extraction and Quantification 6 cells were extracted with 1 ml of ice-cold 60% methanol, followed by centrifugation for 30 min at 16,000 g. The supernatant was immersed at 100°C in a dry bath for 3 min and dried under vacuum. The pellets were dissolved in nuclease-free water for dNTP measurement based on the method described as follows by Ferraro et al. ( Ferraro et al., 2010 Ferraro P.

Franzolin E.

Pontarin G.

Reichard P.

Bianchi V. Quantitation of cellular deoxynucleoside triphosphates. dNTPs from 2 x 10cells were extracted with 1 ml of ice-cold 60% methanol, followed by centrifugation for 30 min at 16,000 g. The supernatant was immersed at 100°C in a dry bath for 3 min and dried under vacuum. The pellets were dissolved in nuclease-free water for dNTP measurement based on the method described as follows by Ferraro et al. (). Briefly, the reaction mixture contained, in a volume of 0.1 ml, 0.1–4 pmol of the dNTP to be determined together with 40 mM Tris-HCl, pH 7.4, 10 mM MgCl 2 , 5 mM dithiothreitol, 0.25 μM oligonucleotide, 1.5 μg RNase A, 0.25 μM labeled dATP, 500–1000 cpm/pmol (or labeled dTTP for the dATP assay) and DNA polymerase. RNase A was included in the assay to remove any labeled RNA formed in metabolic experiments with 3H- labeled nucleosides. The nature and the amount of the DNA polymerase used in the assay are given below for each separate dNTP determination. After 60 min incubation 0.085 ml of the mix was spotted on circular disks of Whatman DE81 paper. After drying, the filters were washed three times for 10 min in large volumes of 5% Na 2 HPO 4 , once in distilled water and once in absolute ethanol. The retained radioactivity was determined by scintillation counting. We used limiting amounts of the Klenow enzyme for dTTP and dATP assays and the Taq DNA polymerase for dCTP and dGTP assays. The chosen enzyme concentration must be balanced by the requirement of a close to linear dose–response curve in the range of the dNTP concentrations to be analyzed. It is advisable to test each new batch of enzyme to find the smallest amount of enzyme that for a given dNTP suffices to give a close to linear standard curve between 0.1 and 4 pmol of the dNTP. We found this range to be optimal for the analysis of dNTPs present in cell extracts. For our Klenow enzyme 0.2 units sufficed for the dTTP assay and 0.025 units for the dATP assay, with 60 min incubation at 37°C. For Taq DNA polymerase 2 units were required for both dCTP and dGTP, with 60 min incubation at 48°C. Once standardized, we used each batch of enzyme for months without change of conditions and without interference by rNTPs.

Analysis of UDP-GlcNAC Using Liquid Chromatography-Mass Spectrometry (LC-MS) For UDP-GlcNAC analysis, a linear ion trap-orbitrap mass spectrometer (Orbitrap Elite, Thermo Fisher Scientific, Bremen, Germany) coupled online with an ultrahigh performance liquid chromatography (UHPLC) system (ACQUITY UPLC, Waters, Millford, MA) was used. The mass spectrometer was operated in the negative ion mode and set to one full FT-MS scan (m/z 150-615, resolution = 15,000) and two FT-MS product ion scans for precursors of UDP-GlcNAC (precursor m/z = 606.08, m/z 150-615, 15,000 resolution) and CTP-13C9 (precursor m/z = 491.03, m/z 300-500, 15,000 resolution). The sample was loaded into the UHPLC system and separated by a HILIC column (BEH Amide, 1.7 μm, 2.1 mm × 100 mm, Waters, Milford, MA), which coupled directly to a mass spectrometer. The solvent A with 20 mM ammonium acetate in 40% ACN with pH = 9 and solvent B with pure ACN were used as the mobile phase for UHPLC separation. The solvent gradient in UHPLC separation was started from 60-99.9% solvent A at 0-3 minutes at a flow rate of 400 μl/min. The total chromatography separation time for each of the analyses was 7 minutes. The fragmentation reactions of m/z 606.08 to 384.99 for UDP-GlcNAC and 491.03 to 393.03 for CTP-13C9 were selected for quantitation using the chromatographic peak area in selected ion chromatogram (SIC). The relative abundance of UDP-GlcNAC was quantified by the abundance of dope CTP-13C 9 .

Cellular RNR Activity Analysis Jong et al., 1998 Jong A.Y.

Yu K.

Zhou B.

Frgala T.

Reynolds C.P.

Yen Y. A simple and sensitive ribonucleotide reductase assay. 6 cells were trypsinized and homogenized in 500 μl of buffer I (50 mM Hepes, pH 7.2; 2 mM DTT, 0.2 mg RNaseA) on ice. After centrifugation at 16,000 g for 15 min, the supernatant was applied onto a Sephadex G-25 spin column, which had been pre-equilibrated with ice-cold buffer II (50 mM Hepes, pH 7.2; 2 mM DTT). The loaded spin column was centrifuged at 1,500 g for 5 min, and the pass-through sample was collected for the RNR activity assay. Protein concentration was measured by using the kit from BioRAD-based Bradford method. Approximately 150 μg protein was used per RNR assay. RNR activity analysis was performed and modified as previously described (). Cells were incubated with growth medium containing the indicated concentrations of glucose for 3 days, and then 2.5 x 10cells were trypsinized and homogenized in 500 μl of buffer I (50 mM Hepes, pH 7.2; 2 mM DTT, 0.2 mg RNaseA) on ice. After centrifugation at 16,000 g for 15 min, the supernatant was applied onto a Sephadex G-25 spin column, which had been pre-equilibrated with ice-cold buffer II (50 mM Hepes, pH 7.2; 2 mM DTT). The loaded spin column was centrifuged at 1,500 g for 5 min, and the pass-through sample was collected for the RNR activity assay. Protein concentration was measured by using the kit from BioRAD-based Bradford method. Approximately 150 μg protein was used per RNR assay. The RNR assay mixture contained 50 mM Hepes, pH 7.2, 6 mM DTT, 4 mM Mg acetate, 2 mM ATP, and 1 μM CDP. The reaction was initiated by adding RNA-free crude extracts and incubated at 37°C for 30 min, and the reaction was terminated at 100°C in a dry bath for 5 min. The substrate CDP was converted to dCDP by RNR and subsequently to dCTP by endogenous NDP kinase in the reaction mixture. After centrifugation at 16,000 g for 5 min, the supernatant was dried under vacuum and dissolved in 16 μl of nuclease-free water for dCTP level determination as described in dNTP quantification. Since RNR activity is the limiting step for dCTP formation in the reaction, the amount of dCTP formation can be represented as RNR activity.

NDPK Activity Analysis Cell crude extracts preparation and enzyme reactions were similar to RNR activity analysis as described in STAR Methods . dCDP instead of CDP was used as the substrate for NDPK activity analysis.

PFK Activity Measurement PFK activity was determined according to manufacturer’s instructions (MAK093, Sigma-Aldrich). In brief, cells were incubated with growth medium containing the indicated concentrations of glucose for 3 or 9 days, and then cells were trypsinized and counted. 2 x 105 cells were homogenized in 200 μl of ice-cold PFK assay buffer. 1/100 dilution of sample was used for subsequent activity assay. PFK activity was reported as nmole/min/mL (milliunit/mL). One unit of PFK is the amount of enzyme that generates 1.0 μmole of NADH per minute at pH 7.2 at 37°C.

sWGA Pull-Down Assay sWGA (succinylated wheat germ agglutinin) is a modified lectin that specifically binds O-GlcNAc on proteins. Pancreatic cells and mammary cells were lysed in Lysis 125 buffer (50 mM Tris, pH 7.4, 125 mM NaCl, 5 mM EDTA, 5 mM EGTA, 0.1% Nonidet P-40, 50 mM NaF, 1 mM PMSF and 1x Proteinase Inhibitor Cocktail (Roche, Indianapolis, IN)). After being clarified by spinning at 15,000 rpm for 15 min, the supernatant was collected and incubated by sWGA beads with or without 0.5 M N-Acetyl-D-Glucosamine (GlcNAc). After overnight incubation at 4°C, the beads were washed three times with Lysis 125 buffer, and sWGA-bound proteins were eluted with Laemmli sample buffer for PFK isoforms and RNR subunits detection by immunoblotting.

Immunoprecipitation and Immunoblotting For RRM1 immunoprecipitation, cells were lysed in RNR lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% NP40, 50 mM NaF, 1 mM Na 2 VO 3 , 1 mM DTT, 0.1% SDS, 2 mM PMSF and protease inhibitors), and the supernatant was incubated with M2 beads at 4°C for 2 hours, or with anti-RRM1 antibody or anti-goat-IgG antibody at 4°C overnight followed by 1-hour incubation with Protein G beads. Beads were washed three times with RNR lysis buffer, and proteins were eluted with Laemmli sample buffer. Protein samples resolved by SDS-PAGE were transferred to PVDF membrane, and then incubated with primary antibody followed by horseradish peroxidase (HRP)-conjugated IgG secondary antibodies (1:5,000, GeneTex). Signals were detected using Luminata Forte Western HRP Substrate (Merk Millipore, WBLUF0500).

Protein Purification pET28a-RRM1, RRM2, or RRM2B plasmid was transformed into E. coli BL21 Tuner (DE3) GST-OGT-WT or GST-OGT-H568A competent cells. The single colony was cultured at 25°C to an OD 600 of 0.6 in LB, and the desired protein was induced with 0.4 mM IPTG at 16°C overnight. Bacteria pellets were resuspended in lysis buffer (50 mM Tris-HCl, pH 7.5 and 250 mM NaCl) and then sonicated with amplitude 80% around 30 sec on ice (Hielscher, Ultrasound Technology). After centrifugation, the supernatant was incubated with Ni-NTA agaroses (Roche, Indianapolis, IN) for 1 hour at 4°C. His-tagged proteins were eluted with 300 mM imidazole, and then imidazole was removed by dialysis.

Protein In Vitro Binding Assay 1 μg of purified His-RRM1 or O-GlcNAc-His-RRM1 was mixed with HPDE or HPNE cell lysates in 0.5 ml of RNR lysis buffer for 1 hour under constant rotation at 4°C. Then Ni-NTA beads were added and incubated for 1 hour at room temperature. Ni-NTA beads (Roche, Indianapolis, IN) were then washed five times, and bound-proteins were analyzed by Western blotting.

Genomic DNA Extraction Tumor-free regions of pancreatic and intestinal tissues from PDAC patients diagnosed with or without DM were collected from paraffin blocks according to pathologist’s recognition. All samples were used for genomic DNA extraction following manufacturer’s instructions (QIAamp DNA Mini kit, 51306).

Amplicon Library Preparation Exon 2, 3, and 4 of KRAS, exon 9 of GAPDH gene, exon 2 of Kras gene and exon 4 of Gapdh gene were used for amplicon amplification and sequencing. The forward and reverse primers that are complementary upstream and downstream of the region of interest were designed with overhang adapters, and used to amplify templates from genomic DNA. A subsequent limited-cycle amplification step was performed to add multiplexing indices and Illumina sequencing adapters. Libraries were normalized and pooled as described by “16S Metagenomic Sequencing Library Preparation”, and sequenced on the MiSeq system.

Sequencing with Illumina MiSeq and Data Analysis The amplicon library was prepared for sequencing according to the manufacturer’s instructions (MiSeq Sequencing protocol). In brief, the amplicon pool was pre-diluted to 2 nM and denatured. 10% of the denatured PhiX control (Illumina, # FC-110-3001) was spiked into the 6 pM amplicon pool and loaded to the sample reservoir of the MiSeq Reagent Cartridge. We used the 500-cycle v2 kit for amplicon sequencing. The run was set by using the MiSeq Control Software (MiSeq Reporter 2.2.29) according to the MiSeq System User Guide Part (# 15027617). A sample sheet (csv-file) for FASTQ format output was designed with the Illumina Experiment Manager 1.5. The run was performed for 2 x 250 bp using a paired-end approach, with automated cluster-generation, Sequencing-by-synthesis (SBS) included reversible terminator sequencing of the indexed samples, and producing demultiplexed FASTQ files. Total 896 bases in the regions of the amplicons including KRAS exon 2-4, total 329 bases in the regions of the amplicons including GAPDH exon 9, total 281 bases in the regions of the amplicons including Kras exon 2, and total 277 bases in the regions of the amplicons including Gapdh exon 4 were counted by bam-readcount ( https://github.com/genome/bam-readcount ). After that, the counts and frequencies of bases at each position in those regions were calculated. The data represent the average of single nucleotide variants.

Lentiviral shRNA, Lentiviral cDNA, and Retroviral cDNA Production for Infection Lentiviral shRNA, lentiviral cDNA, and retroviral cDNA were packaged in 293T and 293GP2 cells, respectively. For lentivirus production, 293T cells (5×106 cells) were co-transfected with 3 μg of pCMVΔ8.91, pMD.G, and 3 μg of pLKO.1-indicatedshRNA or pAS5w-indicated cDNA. For retrovirus production, 293GP2 cells (5×106 cells) were co-transfected with 4 μg of pWZL-Neo-Myr-Flag-PFKL/PFKM) and 4 μg of pMD.G. At 72 h after transfection, culture media containing lentivirus or retrovirus were collected and concentrated on a Millipore concentration column to a final volume of 1 ml. The lentiviral shRNA, lentiviral cDNA, and the retroviral cDNA stocks in 0.2 ml of medium containing 8 μg/ml polybrene were used to infect 2.5×105 cells overnight, after which the media were replaced with complete medium for subsequent assays.

Cell Growth, Cell Cycle, and 2D Clonogenic Cell Survival Analysis Cells plated into a 96-well plate (103 cells/well) were cultured with medium containing the indicated concentrations of glucose and cell growth was measured by the XTT assay (Roche). Cell cycle analysis was performed by staining cells with propidium iodide (PI), which was followed by fluorescence-activated cell sorter (FACS) analysis using CellQuest software (Bd Biosciences). 2D clonogenic cell survival assays were performed by seeding cells at a rate of 500 cells per well into a 6-well plate. After 14 days, the colonies were fixed and stained by Crystal Violet and counted.

Protein Extraction and Western Blotting Analysis Cell extracts were prepared and equal amounts of protein were separated by NuPAGE™ 4-12% Bis-Tris Protein Gels (Thermo Fisher Scientific) followed by electrophoretic transfer to PVDF membranes (Millipore). After blocking with TOOLSpeed Blocking Reagent (TFU-BL500, TOOLS Taiwan) the membrane was incubated with different antibodies overnight at 4°C and treated for 1 hr with horseradish peroxidase-conjugated goat anti-rabbit IgG, goat anti-mouse, and donkey anti-goat antibodies (Santa Cruz). ECL detection of the horseradish peroxidase reaction was performed according to the manufacturer’s instructions (WBLUF0500, Merck Millipore). Protein signal was measured on a UVP BioSpectrum 500 Imaging System.

Cellular ROS Measurement Following culture with the indicated concentrations of glucose for 3 or 9 days, culture media were removed, and cells were loaded with 5 μM H 2 DCFDA diluted in clean medium for 30 min at 37°C. Cells were washed twice with PBS and trypsinized for flow cytometry (FACScalibur, BD Bioscience) analysis using the FL1 channel (Green fluorescence). The profile was examined by CellQuest software (BD Bioscience).

Analysis of NTPs Using Liquid Chromatography-Mass Spectrometry (LC-MS) For NTP analysis, the mass spectrometer was operated in the negative ion mode and set to one full FT-MS scan (m/z 300-600, resolution = 15,000) and nine FT-MS product ion scans (m/z 300-600, 15,000 resolution) for precursors ATP (precursor m/z = 505.99), CTP (precursor m/z = 481.98), UTP (precursor m/z = 482.96), GTP (precursor m/z = 521.99) and CTP-13C9 (precursor m/z = 491.03). The sample was loaded into the UHPLC system and separated by a HILIC column (BEH Amide, 1.7 μm, 2.1 mm × 100 mm, Waters, Milford, MA), which coupled directly to a mass spectrometer. The solvent A with 20 mM ammonium acetate in 40 % ACN with pH = 9 and solvent B with pure ACN were used as the mobile phase for UHPLC separation. The solvent gradient in UHPLC separation was started from 30% solvent A at 0-1 minute, and 30-100% at 1-5 minutes at the flow rate of 400 μl/min. The total chromatography separation time for each of the analyses was 8 minutes. The fragmentation reactions m/z 507.18 to 409.20 for ATP, m/z 481.98 to 384.00 for CTP, m/z 482.96 to 384.99 for UTP, m/z 521.99 to 424.01 for GTP and 491.03 to 393.03 for CTP-13C 9 were selected for quantitation using the chromatographic peak area in selected ion chromatogram (SIC). The relative abundance of NTP was quantified by the abundance of dope CTP-13C 9 .

Cellular NADPH Measurement Cells were harvested after incubation with growth medium containing the indicated concentrations of glucose for 3 days. Cellular NADPH measurement was performed by NADP/NADPH Quantification Kit (MAK038, Sigma-Aldrich) following manufacturer’s instructions.

G6PDH and Aldolase Activity Measurement Cells were harvested for G6PDH and aldolase activity assay according to manufacturer’s instructions (MAK015 and MAK223, respectively, Sigma-Aldrich) after 3 or 9 day-incubation with growth medium containing the indicated concentrations of glucose. G6PDH and aldolase activity were reported as nmole/min/mL (milliunit/mL). One unit of G6PDH or aldolase is the amount of enzyme that generates 1.0 μmole of NADH per minute at pH7.2 at 37°C.

Glucose Uptake Assay 2 x 104 cells per well were seeded into a 96-well plate overnight. Then cells were washed twice with PBS and incubated with 2-NBDG at the indicated concentrations for 10 min or 100 μM 2-NBDG at the indicated time points at 37°C. The reaction was stopped by adding a two-fold volume of ice-cold PBS, and the cells were washed twice with ice-cold PBS. Fluorescence signals before (autofluorescence) and after adding 2-NBDG were measured by using the 485 nm ex and 520 nm em filter set in a multi-well plate reader (Victor-3, Perkin Elmer, Waltham, MA, USA). Cell number per well was determined by Cell Proliferation Kit (XTT assays, Roche, Indianapolis, IN) following manufacturer’s instructions. The glucose uptake, indicated by fluorescent values, was normalized by the cell number. To examine the effect of GLUT and SGLT on glucose uptake in human pancreatic cells, HPDE or HPNE cells were co-incubated with 100 μM 2-NBDG and 10 μM Cytochalasin B or 100 μM Phlorizin in medium containing 5.5 mM glucose. After 10 min, glucose uptake in cells was determined as described above.

Flow Cytometry for Cell Cycle Analysis After high-glucose treatment for 3 days, cells (1 x 106) were fixed in ice-cold 70% ethanol at -20°C overnight. Then, cells were washed with ice-cold PBS once and resuspended in 1 ml of propidium iodide (PI) staining solution (0.1% Triton-x-100, 02 mg/ ml RNasa A and 20 μg/ ml PI). After incubation in dark condition at room temperature for 30 min, the cell cycle profile was examined by flow cytometry analysis (FACScalibur, BD Bioscience) with CellQuest software. For each measurement, data from 10,000 single cell events were collected. For analysis, we first gated on the single cell population using the pulse width vs. pulse area. Then we applied this gate to the scatter plot and gated out the obvious debris. Finally, we combined the gates and applied to the PI histogram plot.

Flow Cytometry for KRASG12D Analysis The cells were fixed and permeabilized with ice-cold methanol (-20°C). Then, cells were incubated with or without the anti-KRASG12D antibody (200 ng / μl, 1:100) for 1 hour at 4°C, followed by staining with Alexa Fluor 488-conjugated secondary antibodies for 1 hour at 4°C. After wash, cells were used for flow cytometry analysis with CellQuest software. Cell debris was removed by gating the major population in a SSC vs FCS density plot.

Flow Cytometry for Glucose Uptake Analysis 5 x 105 cells were seeded in a 60-mm dish overnight. Then 100 μM of 2-NBDG was added and incubated at the indicated time points. After stopping the uptake reaction, cells in each dish were subsequently trypsinized and resuspended in 500 μl ice-cold growth medium for flow cytometry analysis with CellQuest software. Cell debris was removed by gating the major population in a SSC vs FCS density plot. For each measurement, data from 10,000 single cell events were collected.

In-Gel Digestion of Purified RRM1 Protein The purified RRM1 proteins were separated by SDS-PAGE, and the band stained by Coomassie blue were excised, cut into smaller pieces, and destained by 50% acetonitrile (ACN) with 25 mM triethylammonium bicarbonate (TEABC). The proteins in the gel pieces were reduced by 5 mM tris (2-carboxyethyl) phosphine (TCEP) at 37°C for 30 min in the dark and alkylated by 20 mM iodoacetamide (IAM) at 37°C for 60 min in the dark. After extraction with 100% ACN and removing all liquid, the gel pieces were re-saturated with 25 mM TEABC, to which 2 μg trypsin was added, and were incubated at 37°C for 16 h. Digested peptides were extracted by 50% ACN/5% formic acid (FA) twice and 100% ACN and dried completely under vacuum. The peptides were desalted by C18 Zip-tip (Millipore) and subjected to LC-MS/MS analysis.

LC-MS/MS Analysis The desalted peptides were resuspended with 0.1% FA and analyzed by Orbitrap Fusion Tribrid Mass Spectrometer (Thermo Fisher Scientific, San Jose, CA) equipped with a PicoView nanospray interface (New Objective, Woburn, MA). Peptides were loaded onto an analytical C18 column (Acclaim PepMap RSLC, 75 μm i.d. x 25 cm length; Thermo Fisher Scientific) packed with 2 μm particles with a pore size of 100 Å, and were separated using a segmented gradient for 120-min with the following mobile phases: water with 0.1% FA (buffer A) and 2% to 85% ACN with 0.1% FA (buffer B) at 500 nL/min flow rate. Survey scans of peptide precursors from 350 to 1550 m/z with charge states 2-6 were performed at 120K resolution and the AGC target was set to 2 x 105 by Orbitrap. Tandem MS was performed by isolation window at 2.0 Da with the quadrupole, AGC target to 1 x 105, and used for higher-energy collisional dissociation (HCD) fragmentation detected in Orbitrap at a resolution setting of 30K with a normalized collision engergy (NCE) of 26%. Product ion-dependent trigger was set with 204.0867 for HexNAc, 186.075 and 18.065 for HexNAc fragments to filter the O-GlcNAc-conjugated peptides for following MS2 dissociation by electron-transfer dissociation (ETD) or electron-transfer and higher-energy collision dissociation (EThcD). ETD and EThcD were performed at 30K resolution, isolation window at 1.4 Da with the quadrupole, the AGC target was set to 1 x 105 by Orbitrap, and used calibrated charge dependent ETD parameters. Top 20 of given precursors were selectively fragmented and scanned out in mass spectra.

Data Processing and Database Search The raw data were processed by using Proteome Discoverer 2.0 (PD2.0; Thermo Fisher Scientific), and peptide identification was performed by Byonic search engine (version 2.9) against the RRM1 protein sequence with a percolator (strict false discovery rate (FDR) of 0.01 and a relaxed FDR of 0.05). The protease was specified as trypsin with 2 maximum missing cleavage sites. Mass tolerance for precursor ion mass was 10 ppm with the fragment ion tolerance as 20 ppm for HCD, ETD, and EThcD. Oxidation at methionine, carbamidomethyl at cysteine, deamidation at asparagine or glutamine, and HexNAc at serine or threonine were selected as variable modifications. Peptides were considered to be identified if their individual ion score was higher than the identity score (p < 0.05). To evaluate the false discovery rate (< 1%) in protein identification, a decoy database search against a randomized decoy database created by PD2.0 using identical search parameters and validation criteria was also performed. Peptide-spectrum matches (PSMs) with at least high confidence and a strict maximum parsimony principle (target FDR <0.01) were applied for the protein level. All selected MSMS spectra of O-GlcNAc-modified peptides were manually confirmed.