centrifugation (300 X g for 5 min) and dissociated with accucute (Sigma) and the cells were plated at one cell per well in 96-well plate with ultra low attachment. Single cell status was confirmed under microscope. After 12 days incubation, the prostasphere (P2) numbers were counted under a phase contrast microscope. Western blot analysis Total cell lysates from different experiments were obtained by lysing the cells in RIPA buffer containing 50 mM Tris–HCl, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycho- late, 2 mM sodium fluoride, 2 mM Na3VO42, 1 mM EDTA, 1 mM EGTA, and 1 X protease inhibitor cocktail. Western blotting was performed as previously described [47]. siRNA, miRNA and transfection experiments Cells were transfected with 100 nmol/L of Sox2, Nanog, Oct4, Lin28B siRNA or control siRNA (Santa Cruz) as well as 20 nmol/ L of miR-200 or let-7 (Ambion, Austin, TX) using DharmaFECT3 siRNA transfection reagent (DHARMACON, Lafayette, CO). After 3 days of transfection, cells were split and transfected repeatedly with siRNA or miRNA every 3–4 days for indicated times. After 24 h of incubation from last transfection, the cells were collected after trypsinization and then re-suspended in DMEM/F12 (Invitrogen) supplemented with B27 and N2 (Invitrogen). The single cell suspensions were plated on ultra low adherent wells of 6-well plate (Corning) at 2000 cells/well for generation of prostaspheres. In addition, the cell lysates from cells transfected with siRNA or pre-miRNA were prepared for Western blot analysis. Luciferase activity assay PC3 Neo and PC3 PDGF-D cells were seeded at a density of 66103 cells per well in 96-well plate and incubated for 24 h. The cells were co-transfected with Notch1 39UTR luciferase plasmid (Switch Gear Genomics, Menlo Park, CA) and pre-miR-200 using DharmaFECT duo transfection reagent (DHARMACON, Lafay- ette, CO). After 48 h of transfection, luciferase activity was assayed using Steady–Glo Luciferase Assay System (Promega). The renilla luciferase activity was used as a control for transfection efficiency. Real-time RT-PCR The total RNA was isolated using the Trizol reagent. Two micrograms of RNA were transcribed into cDNA using High Capacity RNA-to-cDNA Kit (Applied biosystems, Fostor, CA) according to the manufacturer’s instruction. Real time PCR was used to quantify mRNA expression by using SYBRH Green RT- PCR Reagents (Applied biosystems). Sequences of primers were shown in table S4. The relative amount of RNA was normalized to the expression of GAPDH. For miRNA assay, the total RNA was isolated and PCR was performed as previously described [28]. Microarray for gene profiling and miRNA microarray Microarray and miRNA microarray as well as data analysis were conducted following procedures documented under supple- mentary materials and methods in text S1. All data is MIAME compliant and that the raw data has been deposited in a MIAME compliant database. GEO accession number is GSE22764. Tumor growth in SCID mice Male homozygous CB-17 SCID/SCID mice (8 weeks old) were purchased from Taconic Farms (Germantown, NY). The mice were maintained according to the Animal Care and Use Guidelines approved by the NIH. Mice received Lab Diet 5021 Stem Cell Feature in EMT Cells (Purina Mills, Inc., Richmond, IN). After 1 week of acclimatiza- tion, the mice were randomized into two groups (n = 5) for our study. PC3 Neo and PC3 PDGF-D cells were harvested and re- suspended in serum-free RPMI medium. Cells (16105) in 100 ml serum-free RPMI medium were injected subcutaneously into the right and left flanks of each mouse. As soon as the majority of the tumors began to enlarge (50–75 mm3 volume, as determined by caliper measurements, 15–20th day after cell injection), mice with tumor take rate of 100% were carefully monitored and tumor volume were calculated in each group by twice-weekly caliper measurements. Comparison of growth kinetics between the PC3 Neo and PC3 PDGF-D cell line using this model system, the body weight of mice in each group was also monitored. All mice were euthanized 5 weeks later after the initiation of the experiment because large tumors were formed in the PC3 PDGFD group of mice, which required termination and their final body weight and tumor volume was recorded. On autopsy, the tumor was neatly excised free of any extraneous adhering tissue. The tissue was rapidly frozen in liquid nitrogen, and stored at 270uC and subsequently used for preparation of protein extracts. Total protein was extracted from tumor tissues and prepared for Western blot analysis. Randomly selected harvested tumor tissue from each group of experimental mice were homogenized using Dounce homogenizer (Kontes Co., Vineland, NJ) containing 0.3 ml of ice-cold RIPA buffer solution with added protease inhibitors and 0.1 mM PMSF. The suspension was centrifuged at 16,0006 g at 4uC for 10 min. The supernatant was collected and kept at 270uC until used for Western blot analysis. Immunohistochemistry Formalin-fixed tumor tissue sections were evaluated for tumor cell morphology, mitotic rate, growth pattern, necrosis, cystic change, and inflammatory cellular response. Immunohistochem- ical studies were performed after staining with specific primary antibodies against E-cadherin, Vimentin and Notch1, followed by 3,39-diaminobenzidine (DAB). Sections were visualized under an Olympus microscope (Olympus, Japan) and images were captured with an attached camera linked to a computer. Data analysis Experiments (in vitro) presented in the figures are representative of three or more independent repetitions. The data are presented as the mean values 6 SD. Comparisons between groups were evaluated by a two-tailed student’s t test. Values of p,0.05 were considered statistically significant. Supporting Information Text S1 Supplementary materials and methods. Found at: doi:10.1371/journal.pone.0012445.s001 (0.04 DOC) MB Figure S1 PDGF-D levels in PC3 Neo and PC3 PDGF-D cells. Western Blot showed full length and active form of PDGF-D from PC3 PDGF-D cell lysates (A) and conditioned medium (B). Found at: doi:10.1371/journal.pone.0012445.s002 (1.63 MB TIF) Figure S2 MiR-200b and miR-200c inhibited prostasphere- forming ability of PC3 PDGF-D cells. PC3 PDGF-D cells were transfected with pre-miR-200. 3 days after transfection, cells were split and transfected repeatedly with pre-miR-200 every 3–4 days for 14 days. (A) MiR-200b and miR-200c increased the numbers of prostaspheres with ,100 mm in diameter and decreased the numbers of prostaspheres with .100 mm in diameter. (B) MiR- PLoS ONE | www.plosone.org 12 August 2010 | Volume 5 | Issue 8 | e12445