Introduction 1 2 3 4 5 8 9 10 11 13 14 15 17 15 18 19 20 21 22 Materials and methods Cell cultures l 2 23 Antibodies and chemicals 24 d l 2 Measuring Hcy concentration in growth medium d l l 25 26 d l l d l 27 l t Determination of intracellular S-Adenosylmethionine (SAM) and S-Adenosylhomocysteine (SAH) d l 28 Flow cytometry 23 g 2 Detection of caspase-3 activity Cells were grown in a 96−wells plate (20,000 cells/well). After treatment with Hcy and/or Z-VAD FMK, cells were lysed and incubated with DEVD-rhodamine 110 substrate (Roche, Mannheim, Germany) for 1 h at 37°C. Subsequently the amount of free rhodamine was determined at a microplate fluorescence reader (TECAN spectrafluor, Switzerland). The developed fluorochrome was proportional to the concentration of activated caspase-3 and could be quantified by a calibration curve of diluted free rhodamine. Each condition was measured in triplo per measurement (total of three measurements). Immunofluoresence microscopy To measure the expression of NOX2 and the putative formation of nitrotyrosin, cells were incubated with or without Hcy for 24 h in the 4-well chamber slides (Nalge Nunc International, Naperville, IL, USA). Cells were washed with PBS and fixated with 4% formaldehyde for 10 min at 37°C. Cells were subsequently washed with PBS, permeabilized with acetone–methanol (70%–30%) for 10 min at −20°C, and then washed again with PBS/Tween-20 (0.05% (v/v) Tween-20 in PBS). Subsequently cells were incubated with primary antibodies against NOX2 and nitrotyrosin for 60 min at room temperature followed by incubation overnight at 4°C. PBS and isotype controls were also tested to determine nonspecific binding of the antibodies and background signal. The following day the cells were washed with PBS/Tween and incubated with the secondary antibodies for 30 min at room temperature. After subsequent washes in PBS/Tween and PBS, the slides were covered in mounting medium containing DAPI (Vector Laboratories Inc, Burlingame, CA, USA) to visualize nuclei. Thereafter the slides were covered with coverslips. TM Z TM Ψ Ψ m Ψ m TM Western blot analysis After treatment H9c2 cells were harvested into modified ELB lysis buffer (250 mM NaCl, 0.1% Nonidet P-40, 50 mM HEPES pH 7.0, 5 mM EDTA, 0.5 mM DTT) wherein protease inhibitor cocktail (PIC, 1:40; Sigma) was added, and the cell suspension was mixed rigorously and then incubated for 30 min on ice. After determination of the protein concentration of the samples with the BCA protein assay kit (Pierce, Rockford, IL, USA), reducing sample loading buffer (0.25 M TRIS pH 6.8, sodium dodecyl sulfate (SDS), glycerol, 2-mercaptoethanol, bromophenol blue) was added and the samples were mixed and heated at 95°C for 10 min. A total of 50 μg protein of each sample was then subjected to SDS-PAGE, transferred to nitrocellulose membranes and analyzed for NOX2 expression with monoclonal antibody 48 (1:250 dilution) and for pan-actin with monoclonal antibody 1501R as a loading control (1:6000), followed by horseradish–peroxidase–conjugated rabbit–anti-mouse immunoglobulins (RαM-HRP; 1:1000 dilution; DakoCytomation, Glostrup, Denmark). The blots were visualized by enhanced chemiluminescence (ECL; Amersham Biosciences AB; Uppsala; Sweden). Staining was quantified with a charge-coupled device camera (Fuji Science Imaging Systems; Düsseldorf, Germany) in combination with AIDA Image Analyzer software (Isotopenmessgeräte; Staubenhardt, Germany). ATP measurement via Luciferase–Luciferin assay g g g 2 3 Statistics P Results d l d l l l d l 15 17 26 29 31 d l 1 d l Table 1 d l l t l mM Hcy added mM Hcy measured t t d l l t l t 0.0  0 0.0007 (+/−0.00009) 0.002 (+/− 0.00004) 464.9 (+/− 36.6) 6.3 (+/− 0.7) 0.1  0.14 (+/− 0.03) 0.06 (+/− 0.01)  0.06 (+/− 0.01) 468.3 (+/− 77.2) 7.0 (+/− 2.9) 1.1  1.08 (+/− 0.04) 0.46 (+/− 0.03) 0.32 (+/− 0.01)* 319.6 (+/− 56.2) 16.9 (+/− 6.8) 2.7  2.73 (+/− 0.06) 1.18 (+/− 0.05) 0.84 (+/− 0.03)* ** 164.8 (+/− 34.6)*** n l l  P **  P  P l 25 26 l d l l t l l l P l P 32 34 1 d l d l d l P n P n Effect of Hcy on cell viability d l 1 1 d l P 1 d l 1 d l P P Fig. 1 d l n A B  Caspase-3 activity d l d l P 2 d l Fig. 2 d l n  Effect of Hcy on NOX2 expression and nitrosylation 35 d l 3 d l d l d l P P P 4 d l d l P 4 d l P d l 1 Fig. 3 A B  Fig. 4 A B n  d l P 5 d l P 5 5 Fig. 5 d l A B C D d l n E d l n  ATP depletion 6 d l P d l d l P d l P Fig. 6 d l  Ψ m Ψ m P d l 7 Ψ m d l 7 d l d l 36 Fig. 7 A B d l C d l D d l E Ψ m n  Discussion 8 Fig. 8 A B d l Ψ m  C d l D d l Ψ m.  36 37 38 Ψ m  23 39 40 15 17 41 42 43 Ψ m d l 32 34 d l 44 Conclusion 5 8 15 26 45 48 16 17 49 52 20