fluorescence was read using Microplate Reader (TECAN) at 530/ 590 nm and were photographed. Cell survival assay MTT assay was conducted using AsPC-1, AsPC-1-GTR, MIAPaCa-2, and MIAPaCa-2-GTR as described previously [12] after 72 h of treatment. Combination index and Isobologram for combination treatment were also calculated and plotted using CalcuSyn software (Biosoft, Cambridge, United Kingdom) to determine synergy based on the method of Chou and Talalay [32]. Sphere formation/disintegration assay Single cell suspensions of cells were plated on ultra low adherent wells of 6-well plate at 1,000 cells/well in sphere formation medium [33]. After 7 days, the spheres termed as ‘‘pancreato- spheres’’ were collected by centrifugation and counted [33]. For sphere disintegration assay, 1,000 cells/well were incubated for 10 days, following 5 days of drug treatment, which examined the effect of drug treatment on disintegration of pancreatospheres as described previously [33]. The pancreatospheres were collected by centrifugation and counted under a microscope. Confocal microscopy Single cell suspensions of AsPC-1 and AsPC-1-GTR cells were plated using ultra low adherent wells of 6-well plate at 3,000 cells/ well in sphere formation medium. After 7 days of treatment, the pancreatospheres were collected by centrifugation, washed with 1xPBS, and fixed with 3.7% parformaldehyde. CD44 and EpCAM antibodies were used for immunostaining assay, as described previously [29]. The CD44 or EpCAM-labeled pancreatospheres were photographed by confocal microscopy (Leica TCS SP5) using software LAS AF 1.2.0 Build 4316. Protein extraction and Western blot analysis Proteins were extracted from all four cell lines and also from animal tumor tissues as described previously [12]. Relative level of ABCG2 was evaluated for all four cell lines. The effects on COX- 2, PTEN and b-actin expression were evaluated on tumor tissues by Western blot analysis. as described previously [12]. Animal Experiments The animal protocol was approved by the Animal Investigation Committee, Wayne State University, Detroit, MI. Female CB17 SCID mice 4 wks old were purchased from Taconic Farms (Germantown, NY) and fed Lab Diet 5021 (Purina Mills, Inc., Richmond, IN). Small fragments of the MIAPaCa-2 xenograft were implanted subcutaneously and bilaterally into mice for the drug-efficacy trials. Once the mice developed palpable tumors, they were randomly selected into the following treatment groups References 1. Jemal A, Siegel R, Ward E, Hao Y, Xu J, et al. (2009) Cancer statistics, 2009. CA Cancer J Clin 59: 225–249. 2. Gaviraghi M, Tunici P, Valensin S, Rossi M, Giordano C, et al. (2010) Pancreatic cancer spheres are more than just aggregates of stem marker positive cells. Biosci Rep. 3. Hermann PC, Bhaskar S, Cioffi M, Heeschen C (2010) Cancer stem cells in solid tumors. Semin Cancer Biol 20: 77–84. 4. Ischenko I, Seeliger H, Kleespies A, Angele MK, Eichhorn ME, et al. (2010) Pancreatic cancer stem cells: new understanding of tumorigenesis, clinical implications. Langenbecks Arch Surg 395: 1–10. 5. Lee CJ, Dosch J, Simeone DM (2008) Pancreatic cancer stem cells. J Clin Oncol 26: 2806–2812. 6. Mueller MT, Hermann PC, Heeschen C (2010) Cancer stem cells as new therapeutic target to prevent tumour progression and metastasis. Front Biosci (Elite Ed) 2: 602–613. Targeted Killing of CSCs by CDF (n = 5/group): (1) untreated control; (2) CDF (5 mg/mouse/day), intragastric once daily for 12 days; (3) curcumin (5 mg/mouse/ day), intragastric once daily for 12 days; (4) gemcitabine (1 mg/ mouse/day), intravenous every third day for a total of three doses; (5) CDF and gemcitabine using the doses indicated above; (6) curcumin and gemcitabine using the doses as indicated above. Tumor measurements and changes in weight were performed and tissue was stored at 270uC for RNA and protein extraction. Electrophoretic Mobility Shift Assay (EMSA) assay for assessing the DNA binding activity of NF- kB Nuclear proteins were prepared from tumors tissue using a Dounce homogenizer with 400 ml of ice cold lysis buffer extracted as described earlier [34]. EMSA was performed using the Odyssey Infrared Imaging System with NF-kB IRDye labeled oligonucle- otide from LI-COR, Inc. (Lincoln, NE). Ten mg of the nuclear protein extract was used as described earlier [34]. The NF-kB competition control study was conducted using unlabeled NF-kB consensus oligonucleotide. The samples were loaded and run at 30 mA for 1 hour. The gel was scanned using Odyssey Infrared Imaging System (LI-COR, Inc.). TaqMan miRNA Real-Time Reverse Transcriptase- Polymerase Chain Reaction (RT-PCR) To determine the expression of miRNAs (miRNA-200b, miR- 200c, and miR-21) in MIAPaCa-2 tumors, we used TaqMan MicroRNA Assay kit (Applied Biosystems) following manufactur- er’s protocol. 5 ng of total RNA was reverse transcribed and real- time PCR reactions were carried as described earlier [30], using Smart Cycler II (Cepheid). Data were analyzed using Ct method and were normalized by RNU6B expression. Growth of CSC in xenograft model 5,000 pancreatospheres were isolated and implanted in mice with 1:1 matrigel. The growth rate was observed for a period of 30 days. RNA was extracted from the tumor tissue for subsequent molecular assays as presented under figure legend. Statistical Analysis Comparisons of treatment outcome were tested for statistical difference by the paired t tests. Statistical significance was assumed at a P value of ,0.05. Author Contributions Conceived and designed the experiments: BB SA DK SHS ZW SB AA SP PAP FHS. Performed the experiments: BB SA DK SHS ZW AA. Analyzed the data: BB SA ZW AA FHS. Contributed reagents/materials/analysis tools: PAP SP FHS. Wrote the paper: BB SA FHS. 7. Abas F, Lajis NH, Shaari K, Israf DA, Stanslas J, et al. (2005) A labdane diterpene glucoside from the rhizomes of Curcuma mangga. J Nat Prod 68: 1090–1093. 8. Hatcher H, Planalp R, Cho J, Torti FM, Torti SV (2008) Curcumin: from ancient medicine to current clinical trials. Cell Mol Life Sci 65: 1631–1652. 9. Narayan S (2004) Curcumin, a multi-functional chemopreventive agent, blocks growth of colon cancer cells by targeting beta-catenin-mediated transactivation and cell-cell adhesion pathways. J Mol Histol 35: 301–307. 10. Padhye S, Banerjee S, Chavan D, Pandye S, Swamy KV, et al. (2009) Fluorocurcumins as cyclooxygenase-2 inhibitor: molecular docking, pharmaco- kinetics and tissue distribution in mice. Pharm Res 26: 2438–2445. 11. Padhye S, Yang H, Jamadar A, Cui QC, Chavan D, et al. (2009) New difluoro Knoevenagel condensates of curcumin, their Schiff bases and copper complexes as proteasome inhibitors and apoptosis inducers in cancer cells. Pharm Res 26: 1874–1880. PLoS ONE | www.plosone.org 11 March 2011 | Volume 6 | Issue 3 | e17850