inactivation and/or actin abnormalities indeed underlie the pathology of PKAN, although at present these experiments are hindered by a very limited access to patient-derived material. Surprisingly little is known about the implications of altered de novo CoA synthesis in higher eukaryotes and our results reveal a novel and conserved link between CoA metabolism and the regulation of actin dynamics. We address, for the first time, the consequences of impaired PANK function on actin organization and dynamics and the results obtained contribute to the general cell biology knowledge and may potentially increase our under- standing of the human neurodegenerative disorder PKAN. Materials and Methods Drosophila cell culture - Drosophila Schneider’s S2 cells were maintained at 25uC in Schneider’s Drosophila medium (Invitrogen) supplemented with 10% heat inactivated fetal calf serum (Gibco) and antibiotics (penicillin/streptomycin, Invitro- gen). Cells in the exponential phase of growth were used for all the experiments. When required 0,5 mM HoPan (Zhou Fang Pharm Chemical, Shanghai, China) and/or 0,1 mM pantethine were added directly to the cell culture medium. HoPan treatment was continued for 2–3 days. dsRNA synthesis and RNAi T7-flanked primer sequences were designed to amplify gene specific DNA templates (for primer sequences, see Table S1). DNA templates were amplified using standard PCR methods. Double-stranded RNAs were obtained by in vitro transcription and purified using MegaScript RNAi Kit (Ambion). 2 pmols of DNA templates were used per 20 mL in vitro transcription reaction. dsRNA treatment was carried out as described previously [54]. Cells were incubated in serum-free medium containing 40 nM dsRNA for 1 hour, following the addition of serum containing medium. The cells were incubated for 4 days to induce an efficient knock-down. Cells were subcultured, drugs (HoPan and/or pantethine) were added to the medium and the cells were maintained for additional 2–3 days until analysis. SHSY-5Y culturing and differentiation SHSY-5Y human neuroblastoma cells were maintained in dMEM (Invitrogen) supplemented with 10% FCS (Gibco) and antibiotics (penicillin/streptomycin, Invitrogen). Cells of a passage between 10–30 were used. For HoPan treatment, cells were cultured in custom made dMEM without vitamin B5 (Thermo Scientific) supplemented with dialyzed FCS (Thermo Scientific). This medium did not affect normal growth of the cells. Cells were differentiated with 50 mM retinoic acid (RA, Sigma) for 48– 72 hours. Differentiated SHSY-5Y cells were plated in poly-Lysine (for this poly-D or poly-L-lysine was used, resulting in an identical outcome) coated culture dishes and cell morphology and neurite formation were quantified after 48 hours respectively. Neurite outgrowth was quantified as a percentage of highly b-III-tubulin positive cells bearing at least one projection exceeding two lengths of the cell body. Immunoblotting Equal numbers of Drosophila S2 cells were centrifuged, washed once with PBS and lysed immediately in 16 Laemmli Sample Buffer (62,5 mM Tris/HCl pH 6,8; 2% SDS; 10% glycerol; bromophenol blue) with 10 mM NaF. SHSY-5Y cells were washed 1 time with PBS in tissue culture dishes and removed Coenzyme A Levels Influence Phospo-Twinstar Levels from the surface by scraping the cells into PBS. After centrifuga- tion cells were lysed in 16 Laemmli Sample Buffer with 10 mM NaF. All sample were further sonicated and boiled for 4 min with 5% b-mercaptoethanol. Protein extracts were run on 12,5% SDS- Page gels, transferred onto nitrocellulose membranes and blocked with 5% milk in PBS/0,1% Tween, following by an overnight incubation with primary antibodies. The following antibodies were used: rabbit anti-dPANK/Fbl [5], rabbit anti-Twinstar [20], rabbit anti-cofilin and rabbit anti-phospho-cofilin (Cell Signaling), rabbit anti-phosphorylated AMP-activated kinase (Cell Signaling #2535) and mouse anti-tubulin (Sigma). HRP-conjugated sec- ondary antibodies (Amersham) were used and detection was performed using enhanced chemiluminescence. Band intensities were quantified with Adobe Photoshop. Immunofluorescence For immunofluorescence Drosophila S2 cells were seeded on concanavalin A-coated glass microscope slides and allowed to spread for 45 min. SHSY-5Y cells were plated on poly- lysine- coated glass slides (for this poly-D or poly-L-lysine was used, resulting in an identical outcome) and cultured for 48 hours. Cells were fixed with 3,7% formaldehyde for 15 minutes, washed 3 times with PBS and permeabilized with 0,2% Triton X-100 for 10 minutes. Slides were further blocked with 3% BSA in PBS/ 0,1% Tween for 30 minutes, followed by the 2 hour-incubation with primary antibody (mouse anti-beta-III-tubulin (clone 2G10, Sigma)). Secondary goat anti-mouse-Alexa488 antibodies (Molec- ular Probes) were used according to the manufacturer instructions (1 hour incubation). To visualize filamentous actin, cells were stained for 20 minutes with 20 U/mL rhodamine phalloidin (Molecular Probes) diluted with 3% BSA in PBS/0,1% Tween. Finally, microscope slides were mounted using CitiFluor mounting medium (Citifluor Ltd). Fluorescent microscopy, live cell imaging and image quantification Fluorescent images were obtained with a Leica fluorescent microscope and processed with Leica software and Adobe Photoshop. More than 150 cells (from at least 10 different microscopic fields) per condition were counted to quantify the cell spreading (S2 cells) and neurite formation (SHSY-5Y cells). Phase contrast live cell imaging was performed on a Zeiss 780 confocal microscope with a 636 glycerol immersion lens. S2 cells were seeded in ConA coated glass bottom culture dishes MatTek Corporation), imaged for 20 minutes per movie in 20 second intervals. Images were processed and kymographs created with ImageJ. For this, squares of 5–10 pixels thickness were drawn across the cell edges and the individual images combined to generate the kymographs. Representative cells, movies and kymographs are shown for each experiment. Statistical analysis All results were obtained in at least two independent experi- ments, with a duplo or triplo of each experimental condition. Statistical significance was calculated using the Student’s t-test (two-tailed, with equal variance). p values,0,05 were considered significant and were indicated as follows: p,0,05 - *; p,0,01 - **; p,0,001 - ***. Graph error bars represent S.E.M. Supporting Information Figure S1 Inhibition of dPANK activity in S2 cells induces an increase in phospho-Twinstar levels after 4 days of RNAi treatment. (A) S2 cells were treated with control PLOS ONE | www.plosone.org 7 August 2012 | Volume 7 | Issue 8 | e43145