Materials

4.1.1. Cell line and cell culture reagents

MIN6 cells were originally obtained from Professor Jun-ichi Miyazaki (Osaka Medical School, Japan). Dulbecco’s Modified Eagles Medium (DMEM), CO2-independent medium and 0.25% Trypsin-EDTA solution were purchased from Invitrogen Life Technologies (Paisley, UK) and Fetal Bovine Serum (FBS) from Biosera (Boussens, France). Penicillin-Streptomycin solution (10,000 units penicillin and 10 mg streptomycin per mL in 0.9% sodium chloride), trypan blue and thiazolyl blue tetrazolium bromide (MTT) were all purchased from Sigma Aldrich (Gillingham, Dorset, UK). Trypsin neutralising solution (0.05% trypsin inhibitor, 0.1% BSA) was from Promocell (Heidelberg, Germany). Neubauer haemocytometers for cell counting were purchased from Paul Marienfeld GmbH & Co. KG (Lauda-Königshofen,Germany).

4.1.2. Enzymes, peptides and proteins

L-Arginyl-glycyl-L-aspartic acid (RGD), human fibronectin purified from plasma and human placental collagen IV were obtained from Sigma Aldrich and mouse Engelbreth-Holm-Swarm (EHS) lathrytic tumour derived collagen IV was obtained from BD Bioscience (San Jose, USA). Collagenase from Clostridium histolyticum (type VII), pronase E from Streptomyces griseus (type XIV), pepsin from porcine gastric mucosa, prolidase from porcine kidney, leucine aminopeptidase from kidney microsomes (type VI-S), L-lactic dehydrogenase from rabbit muscle and D-lactic dehydrogenase from Staphylococcus epidermidis were all purchased from Sigma Aldrich.

4.1.3. Antibodies

Armenian hamster anti-mouse Itgb1 (functional grade purified) monoclonal antibody was from eBioscience (Hatfield, UK). Chicken polyclonal anti-insulin antibody and rabbit polyclonal anti-collagen IV antibodies were obtained from Abcam (Cambridge, UK). Mouse anti-MG-H1 monoclonal antibody clone 1H7G5 was a gift from Professor Michael Brownlee (Albert Einstein College of Medicine,

83 Bronx, NY). Alexa Fluor 555 goat anti-chicken IgG, Alexa Fluor 488 goat anti- mouse IgG, Alexa Fluor 488 goat anti-rabbit IgG and Alexa Fluor 555 goat anti- rabbit IgG were all obtained from Molecular Probes (Invitrogen, Eugene, Oregon, USA).

4.1.4. Analytical and preparative kits

RNeasy Mini Kit was from Qiagen (Manchester, UK). CBQCA protein quantitation kit was purchased from Invitrogen Life Technologies (Paisley, UK) and the protein assay dye concentrate from BioRad (Hemel Hempstead, Hertfordshire, UK). Glucose assay kits were obtained from Sigma Aldrich. Strong anion exchange solid phase extraction cartridges were from Alltech Associates Ltd. (Carnworth, Lancashire, UK) and 3,000 and 10,000 molecular weight cut-off centrifugal filters were from Millipore (Watford, UK).

4.1.5. Immunohistochemistry reagents

Microscope slides were from Fisher Scientific (Loughborough, UK) and cover glass from VWR International (Lutterworth, Leicestershire, UK). Embedding matrix was from Shandon Cryomatrix (ThermoFisher, Epsom, Surrey, UK). Mounting medium containing 4’,6-diamidino-2-phenylindole (DAPI) and an ImmEdge hydrophobic barrier pen were purchased from Vector Laboratories (Peterborough, UK).

4.1.6. Chromatographic reagents

Hypercarb columns (50 x 2.1 mm, 150 x 2.1 mm and 250 x 2.1 mm) and octadecyl silane (ODS) columns (100 x 2.1 mm) for HPLC were purchased from Thermofisher (Epsom, Surrey, UK) and Waters (Elstree, Hertfordshire, UK) respectively. HPLC grade and Optima grade solvents were obtained from Fisher Scientific (Loughborough, UK).

4.1.7. Other analytical reagents

Nuclease-free water and 18S reference primers were from Qiagen (Manchester, UK). All other primers were ordered from Invitrogen custom primer service (Paisley, UK). dNTP mix was from Fermentas (Thermo Scientific, Epsom, Surrey, UK). Bioscript, RiboSafe RNase Inhibitor, Oligo (dT)18 Primer Mix and

84 SensiMix Sybr Low-ROX were all purchased from Bioline (London, UK). [glycine- 13

C215N]GSH, 98% 15N and 99% 13C was purchased from Sigma Aldrich. [13C415N2]GSSG was synthesised from [glycine-13C215N]GSH. Standards and isotopic standards for dicarbonyl assay were available in house. 3-DG and [13C6]3- DG were synthesised from glucose and [13C6]glucose respectively. Glyoxal and [13C2]glyoxal were synthesised by oxidation of ethylene glycol and [13C2]ethylene glycol respectively using alcohol oxidase and catalase. High purity methylglyoxal and [13C2]methylglyoxal were available in house, prepared and purified as described (McLellan and Thornalley, 1992). Isotopically labelled citrulline and amino acids were purchased from Cambridge Isotope Laboratories (Andover, MA, USA) and glycation, oxidation and nitration adducts were available in house, prepared and purified as described (Ahmed, et al., 2002; Thornalley, et al., 2003). 2,5,5[2H3]DL- α-Aminoadipic acid ([2

H3]-AAA) was purchased from CDN Isotopes (Quebec, Canada). ε-(γ-L-Glutamyl)–L-lysine was purchased from Bachem AG (Bubendorf, Switzerland) and [13C5]ε-(γ-L-glutamyl)–L-lysine was available in house, prepared and purified as described (Bertholet, et al., 1977). S-p-Bromobenzyl-glutathione cyclopentyl diester (BrBzGSHCp2) was available in house, prepared and purified as described (Thornalley, et al., 1996). All other analytical reagents and buffers were purchased from either Sigma Aldrich or Fisher Scientific.

4.1.8. Instrumentation

Water used for all experiments was filtered through a Milli-Q Advantage A- 10 System from Millipore (Watford, UK). Microplate assays were quantified using a FLUOstar Optima plate reader (BMG Labtech, Aylesbury, Buckinghamshire, UK) and enzymatic activity assays and stock calibrations performed using UVIKON XS spectrophotometer (NorthStar Scientific Ltd. Potton, Bedfordshire, UK). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was performed using Waters Acquity ultra HPLC systems with a Quattro Premier XE Tandem mass spectrometer and Xevo triple quadrupole mass spectrometer (Waters, Elstree, Hertfordshire, UK). Enzymatic hydrolysis was performed using a CTC-PAL Automation System (CTC-Analytics, Zwingen, Switzerland). The centrifugal evaporator was a Savant Instruments SpeedVac (Thermo Scientific, Waltham MA). Quantification of RNA stocks was performed using a ND-1000 Nanodrop spectrophotometer (Nanodrop, Wilmington, USA). RNA expression analysis was

85 performed using a 7500 Fast Real-Time PCR machine from Applied Biosystems (Carlsbad, California, USA). A Sonics Vibra-Cell sonicator from Jencons Scientific (Leighton Buzzard, Bedfordshire, UK) was used for lysis of cell culture samples and a Lab Gen 7 homogeniser from Cole Parmer (Vernon Hills, USA) was used for isolation of protein and soluble extracts from pancreatic tissue samples. Pancreas samples were sectioned for immunohistochemistry using a CM1850 Cryostat from Leica Microsystems (Milton Keynes, UK). Confocal microscopy was performed using an AxioVert 200M laser scanning confocal microscope from Carl Zeiss Microscopy (Thornwood, NY, USA). AFM-FS was performed using a CellHesion module (JPK Instruments, Berlin, Germany).

4.1.9. Software

Mass spectrometry data was processed using MassLynx software Version 4.1 (Waters, Elstree, Hertfordshire, UK). Atomic force spectroscopy data was processed using CellHesion 200 software (JPK Instruments, Berlin, Germany). Confocal microscopy images were viewed using LSM Image Browser (Carl Zeiss Microscopy, Thornwood, NY, USA). Microplate assays were analysed using Optima software version 2.10 R2 (BMG Labtech, Aylesbury, Buckinghamshire, UK). Real-time PCR analysis was performed using 7500 Fast System Software version 1.4.0. RNA primers were designed using the Oligo perfect design tool (Invitrogen Life Technologies, Paisley, UK) with mRNA and genomic sequences obtained using the University of California, Santa Cruz genome browser tool (Genome Bioinformatics Group, University of California, Santa Cruz). ChemDraw Pro version 13.0 was used for chemical structures and associated analysis. Statistical analysis was performed using SPSS Statistics version 21.