ylated Chk2 was detected by using 1:50 diluted rabbit monoclonal antiChk2-P antibody for 1 h. Detection of cleaved PARP was performed by incubation with 1:50 diluted Alexa Fluor 488-conjugated rabbit monoclonal anti-cleaved PARP antibody. Following secondary antibodies were: 1:500 diluted Alexa Fluor 633 F2 fragment of goat anti-mouse IgG , 1:100 diluted FITC goat anti-mouse IgG or 1:100 diluted FITC goat antirabbit IgG for 45 min. All incubation steps were performed on ice. Cells were PF-562271 analysed on FACSCalibur using CellQuest Pro or FlowJo software. For each sample 10,000 cells were counted. were collected. Cellular aggregates and debris were excluded from analysis by proper gating. Data were fit to define the G1, S, and G2/M phases by using the Dean-Jett-Fox mathematical model of the FlowJo software. Mitochondrial cytochrome c release At 24 h post transfection, HeLa cells were trypsinized and investigated for cytochrome c release by using the FlowCellect cytochrome c kit from Millipore according to manufacturer’s instructions. Cells were analysed with FACSCalibur using Cell Quest Pro or FlowJo software. For each sample 10,000 cells were counted. 
Western blotting Twenty-four hours post transfection, HeLa cells were homogenized in ice-cold RIPA buffer containing protease inhibitors, PMSF and sodium orthovanadate. The homogenate was clarified by centrifugation and the total protein amount of supernatant was determined by using the Bradford method. Equal amounts of proteins were mixed with LDS Sample Buffer and Sample Reducing Agent, heated for 10 min at 95C and were subjected to 4-12% Bis-Tris Gel at 125 V in MES SDS Running Buffer. The bands were electrotransferred to nitrocellulose membranes in Transfer Buffer for 1 h at 100 mA. Membranes were blocked for 1 h with 5% non-fat dry milk 22988107 in PBS containing 0.1% Tween-20 . Membranes were incubated overnight at 4C with primary antibodies. These were: 1:500 diluted rabbit monoclonal anti-cleaved caspase-3 antibody and 1:1000 diluted rabbit monoclonal anti-tubulin antibody. All antibodies were diluted in 5% non-fat dry milk with PBST. After incubation with the respective 1:2000 diluted goat horseradish peroxidase-conjugated secondary antibody, membranes were subjected to detection by ECL detection Supersignal West Pico Chemiluminescent Substrate. For the detection of-actin 1:25000 diluted anti-actin antibody cross-linked to horseradish peroxidase was used. For the detection of V5 1:5000 diluted anti-V5 antibody cross-linked to horseradish peroxidase was applied. ImageStream analysis At 24 h post transfection HeLa cells were fixed, permeabilized and stained as described for flow cytometry. After staining of nuclei with DAPI, cells were analyzed on an ImageStream multispectral flow cytometer and images were analyzed using IDEAS image-analysis software. Ten thousand events were collected in each sample and single stained controls were used to compensate fluorescence between channel images on a pixel-by-pixel basis. The instrument combines the features of classic fluorescence microscopy and flow cytometry so allowing multiparametric analyses. The machine enabled gating around single cells, allowing detailed morphological analysis based on acquired cellular images. 12176911 Nuclear translocation of A3A was determined by using the similarity feature in the IDEAS software. The similarity score provides a measure of the degree of nuclear localization of A3A by computing the pixel intensity correlation between the