After washing

the cells 3 × 5 min with 500 ul cold PBS, Selleck BIRB 796 the cells were permeabilized with 0.5% Triton X-100 in PBS for 2 min. Slides were washed 3 × 5 min with cold PBS and then blocked with PBS containing 2% BSA (w/v) for 60 min. The following primary antibodies were used for both cell lines: mouse anti-c-Myc 9E10 (Santa Cruz), Cell Cycle inhibitor dilution 1:300; rabbit anti-TbV-H+PPase (visualization of acidocalcisomes, a gift of Théo Baltz, University of Bordeaux II, France; dilution 1:500); Secondary antibodies were Alexa Fluor 488 or 594 conjugated goat anti-mouse or goat anti-rabbit (Molecular Probes; highly cross-absorbed, dilution 1:750). DAPI-staining was done with Vectashield mounting medium with DAPI (Vector Laboratories). Coverslips were mounted with Vectashield mounting medium containing DAPI (Vector Laboratories) and images were obtained using a LEICA DM 6000B microscope. Hypoosmotic treatment Wild-type cells and knock-out

clones were subjected to hypoosmotic treatment using a published procedure [28]. Briefly, exponentially growing cultures were centrifuged for 5 min at 3000 rpm. Individual cell pellets were suspended in PBS diluted with H2O to 1×, 0.8× and 0.4× regular strength, and were incubated at 27°C for 30 min. Cells were then collected by centrifugation for 10 min at 2,500 rpm, resuspended in regular SDM-79 medium and their density was adjusted to 2 × 106 cells/ml. Cell density was again CBL-0137 datasheet determined and slides for immunofluorescence Cyclooxygenase (COX) were prepared after 24 h incubation. ATP determination For the determination of intracellular ATP, triplicate aliquots of 5 × 106 cells were

centrifuged for 5 min at 6000 rpm. The cell pellet was suspended with 150 μl cold 1.4% perchloric acid. After incubation for 30 min on ice, 30 μl of 1N KOH were added. After incubation on ice for an additional hour, samples were centrifuged for 20 min at 13,500 rpm. 150 μl of the resulting supernatant were withdrawn for further analysis. 10 μl aliquots of such supernatant were then analyzed using the ATP Bioluminescence Assay Kit CLS II (Roche) according to the instructions of the supplier. To calculate intracellular ATP concentrations, cell volumes of 96 ± 8 μm3 (9.6 × 10-14 l) for procyclics and 53 ± 3 μm3 (5.3 × 10-14 l) for the bloodstream form (Markus Engstler, University of Würzburg, FRG; personal communication) were assumed. Polyphosphate determination Total cellular polyphosphate was determined according to published procedures [29, 30]. Cells (2 – 5 × 106) were centrifuged, the supernatant was carefully withdrawn and the cell pellets were snap-frozen and stored at – 70°C. Polyphosphates were extracted by incubating the cell pellets with 1 ml HE buffer (25 mM HEPES, pH 7.6, 1 mM EDTA) for 30 min at 85°C, with intermittent vortexing.