Synthesis and packaging of retroviral constructs Optimal short hairpin RNA 19mer sequences targeted to human PINK1 (NM_032409) were determined using the Dharmacon siDESIGN Center (http://www.dharmacon.com). The 19mers with the highest predicted efficacy (scores $7) were selected. Single stranded 64mers were obtained from Operon. The forward and reverse sequences are detailed in supplementary Methods S1 online. Oligomers were annealed and ligated into pSUPER.retro.puro (PSR) mammalian expression vector (Oligoengine) according to the manufacturer’s protocol. Ligation mixtures were used to transform JS4 competent E.coli. Maxipreps of successfully ligated vectors were prepared using a Quaigen kit and sequenced. Vectors were packaged into retroviral particles using y-NX-Ampho cells. Viral supernatant harvested from transfected cells was sterile filtered and stored at -70uC until required. Production of stable cell lines Early passage NSCs were infected with viral supernatant containing PSR vector, PSR containing shRNA sequences (supple- mentary Methods S1 online). When confluent, NSCs were split and transduced cells selected for using Puromycin (1 ug/ml). Cell colonies were picked and further propagated before screening by RT-PCR. Immunofluorescence and image acquisition Immunostaining of coverslips and 96 well plates was carried out as described previously [39]. Details of all antibodies are available in supplementary data online (Table S1). For live dye staining of organelles, Mitotracker Red CMXRos or Lysotracker was used according to manufacturer’s instructions. Coverslips were mount- ed onto slides using fluorescent mounting medium (Dakocytoma- tion). Non-confocal images were obtained using an Axioplan 2 MOT microscope (Zeiss) with filters for FITC, Rhodamine and DAPI and Plan Neofluar 25x/0.8 Ph2 objective at RT. Images were taken using an AxioCam MRm (Zeiss) camera and controlled using the Axiovision Control software. Alternatively, images were obtained using an LSM510 META confocal microscopy system (Zeiss) equipped with ‘‘plan-Apochromat’’ 63x/1.40 Oil DIC objective at RT controlled by Zeiss LSM software. Fluorescence was recorded at 488nm using 30mW Ar- laser for excitation or at 543 nm using 1mW HeNE laser for excitation. Zeiss Immersol was used as imaging medium. For counting of tyrosine hydroxylase (TH) and neurofilament (NF) positive cells, the Cellomics ArrayScan HCS reader was used and images from 30 fields of view per well were analysed using the Target Activation BioApplication software. Transmission Electron Microscopy Human NSCs were plated on 13mm Thermanox coverslips coated with laminin. When confluent, NSCs were differentiated using the SD/DA protocol for 47 dd and fixed using 3% glutaraldehyde in 0.1 M sodium cacodylate buffer and 5 mM CaCl2, pH 7.4. Specimens were dehydrated through a graded series of Analar Ethanol (70% 90% 100%). Cells were embedded in araldite resin mixture and sectioned using a Richert Ultracut S microtome. Sections were stained using 25% Uranyl acetate in methanol and Reynold’s Lead Citrate and images were taken on a Philips CM10 transmission electron microscope at 3800X and 9800X magnification. Images were taken using the KeenView system from SIS, resized and annotated in Adobe Photoshop. Live cell imaging Live cell imaging was performed as described previously [41]. See supplementary Methods S1 online. Briefly, cells were PINK1 Deficiency bathed at RT in a HEPES-buffered salt solution (HBSS) containing (mM): 156 NaCl, 3 KCl, 2MgSO4, 1.25 KH2PO4, 2 CaCl2, 10 glucose and 10 HEPES, pH 7.35. For Dym measurements, 25 nM TMRM was included and cells were incubated for 45 min. Z-stacks of .20 neurons per clone were obtained using a Zeiss LSM confocal microscope. Images were analysed using Lucida 6 software. For ROS measurements, MitoSOX (10 mM) was loaded for 10 min followed by washing. Dihydroethidium (HEt, 10 mM) was present in all solutions throughout the experiments. Confocal images were obtained using a Zeiss 510 uv-vis CLSM equipped with a META detection system and a 40x oil immersion objective. HEt or MitoSOX were excited using the 543 nm laser line and fluorescence measured using a 560nm longpass filter. Measurement of total and reduced glutathione. See supplementary Methods S1 online Citrate synthase activities Statistical analyses All experiments were performed with three different cell clones, in triplicate. Data were analysed by parametric Student T-tests and significance expressed as follows *P,0.05, **P,0.01, ***P,0.001 unless otherwise stated. For all graphs bars represent mean6SEM. Online supplementary material Generation of PINK1 knockout mice Primary embryonic mouse cultures Table S1 Antibodies SDS-PAGE and immunoblotting Production of stable cell lines Measurement of total and reduced glutathione Live cell imaging ETC activities Results PINK1 is up-regulated following differentiation of human NSCs to dopaminergic neurons We first sought to validate the use of the human NSC model, differentiated to human midbrain neurons, as a useful model to test PINK1 function. RT-PCR confirmed upregulation of expression of a range of dopaminergic neuronal markers (tyrosine hydroxylase, TH, dopamine transporter, DAT) upon differentiation of NSCs to human neurons (Figure S1A). In addition there was an upregula- tion of the expression of markers Lmx1a and Nurr1, both of which are involved in committing precursor cells to dopaminergic differentiation. Furthermore we demonstrated that the human neurons expressing TH, also expressed PINK1. PINK1 expression is highest in mature neurons as demonstrated by immunofluores- cence (Figure 1A). PINK1 was detected by RT-PCR in human NSCs (Figure 1C). Moreover, PINK1 expression was found to increase up to ,100 fold following differentiation to neurons, (Figure 1C) suggesting a physiological role of PINK1 within differentiated human neurons. This high level of PINK1 expression in differentiated human DAergic neurons validated the use of this model in examining the effects of loss of PINK1 function. RNAi-mediated knockdown of PINK1 mRNA in human cell lines Using retroviral mediated PINK1 shRNA expression, levels of PINK1 knockdown ranging from 10–90% were achieved. The efficiency of PINK1 knockdown (PINK1 kd), determined by RT- PCR, was found to be shRNA sequence specific, with sequence PLoS ONE | www.plosone.org 4 June 2008 | Volume 3 | Issue 6 | e2455