Same blot. B. LNCaP cells were transfected with 100 nM siRNA targeting Luc or TCTP, seeded in 10 cm Otein. For the PAP4 serum that did not produce significant matches plates and assayed for colony formation ability by crystal violet staining after three 10781694 weeks of growth. Representative pictures are shown. C. Quantification of colony formation of LNCaP cells after siRNA mediated knockdown of TCTP. The graph represents two experiments in triplicate. The differences between the groups were evaluated using two-tailed, paired Student’s t-test compared to Luc-siRNA treated samples, with *: P,0.05 indicating significant difference. doi:10.1371/journal.pone.0069398.gminimize suffering were according to IACUC guidelines and have previously been described [22,23].additional 24 h in RPMI 1640 containing 0.5 CT-FCS prior to treatment with 1028 M R1881 (synthetic androgen). R1881 was obtained from NEN.Cell CultureLNCaP cells were obtained from the American Type Culture Collection (ATCC) and cultured in RPMI 1640 medium supplemented with 10 Fetal Calf Serum (FCS), 2 mM Lglutamine, 50 U/ml penicillin and 50 mg/ml streptomycin (Lonza). The human bronchial epithelial BEAS-2B cell line transformed with SV40 was a generous gift from Sten Mollerup (Department of Toxicology, National Institute of Occupational Health, Oslo, Norway) [24]. BEAS-2B cells were maintained in LHC-9 medium supplemented with 1.8 mg/ml bovine serum albumin (BSA) on plates coated with 0.01 mg/ml fibronectin and 0.03 mg/ml bovine collagen dissolved in PBS. For both cell lines the culture media was changed every two to three days, and the cells were incubated at 37uC in a humidified 5 CO2, 95 air incubator. For androgen induction experiments, LNCaP cells were starved for 48 h in RPMI 1640 containing 2 charcoal treated (CT)-FCS, followed by anXenograft ExperimentsTransplantation, growth, and harvest of tumors from mice bearing CWR22 xenografts were as described previously [22,23]. CWR22 xenografts were grown in nude mice in the presence of a sustained-release testosterone pellet. After tumor growth, mice were castrated, the testosterone pellets were removed and the regressing tumors were collected at 1, 2, or 4 weeks after castration. Animals were housed and treated according to the IACUC guidelines.Quantitative PCR (qPCR)Upon harvest, total RNA was extracted from cells using TrizolH reagent (Invitrogen) according to manufacturer’s recommendations. 1? mg RNA was used for first-strand cDNA synthesis with the SuperScript II system (Invitrogen) and oligo-dT primers. qPCR analyses were performed on the LightCyclerTCTP in Prostate CancerFigure 3. Downregulation of TCTP expression leads to increased apoptosis in LNCaP cells. LNCaP cells were cultured on coverslips and transfected with siRNA against TCTP or Luciferase (Luc) for 72 h. Apoptosis was induced by 100 nM TG for 36 h during siRNA treatment. After treatment and fixation, apoptotic cell death was detected using the TUNEL assay, cell nuclei were stained with DAPI and cells were visualized using Axioplan imaging microscope. A. Representative pictures of LNCaP cells transfected with either TCTP- or Luc-siRNA for 72 h and treated with DSMO or TG for 36 h during transfection. TUNEL positive cells appear as red spots. Arrows indicate apoptotic cells. B. Quantification of apoptosis incidence. The data show the percentage of nonviable cells after 36 h treatment with TG in cells transfected with TCTP or Luc siRNA. Columns represent the mean of three independent experiments performed in Title Loaded From File triplicate and bars.Same blot. B. LNCaP cells were transfected with 100 nM siRNA targeting Luc or TCTP, seeded in 10 cm plates and assayed for colony formation ability by crystal violet staining after three 10781694 weeks of growth. Representative pictures are shown. C. Quantification of colony formation of LNCaP cells after siRNA mediated knockdown of TCTP. The graph represents two experiments in triplicate. The differences between the groups were evaluated using two-tailed, paired Student’s t-test compared to Luc-siRNA treated samples, with *: P,0.05 indicating significant difference. doi:10.1371/journal.pone.0069398.gminimize suffering were according to IACUC guidelines and have previously been described [22,23].additional 24 h in RPMI 1640 containing 0.5 CT-FCS prior to treatment with 1028 M R1881 (synthetic androgen). R1881 was obtained from NEN.Cell CultureLNCaP cells were obtained from the American Type Culture Collection (ATCC) and cultured in RPMI 1640 medium supplemented with 10 Fetal Calf Serum (FCS), 2 mM Lglutamine, 50 U/ml penicillin and 50 mg/ml streptomycin (Lonza). The human bronchial epithelial BEAS-2B cell line transformed with SV40 was a generous gift from Sten Mollerup (Department of Toxicology, National Institute of Occupational Health, Oslo, Norway) [24]. BEAS-2B cells were maintained in LHC-9 medium supplemented with 1.8 mg/ml bovine serum albumin (BSA) on plates coated with 0.01 mg/ml fibronectin and 0.03 mg/ml bovine collagen dissolved in PBS. For both cell lines the culture media was changed every two to three days, and the cells were incubated at 37uC in a humidified 5 CO2, 95 air incubator. For androgen induction experiments, LNCaP cells were starved for 48 h in RPMI 1640 containing 2 charcoal treated (CT)-FCS, followed by anXenograft ExperimentsTransplantation, growth, and harvest of tumors from mice bearing CWR22 xenografts were as described previously [22,23]. CWR22 xenografts were grown in nude mice in the presence of a sustained-release testosterone pellet. After tumor growth, mice were castrated, the testosterone pellets were removed and the regressing tumors were collected at 1, 2, or 4 weeks after castration. Animals were housed and treated according to the IACUC guidelines.Quantitative PCR (qPCR)Upon harvest, total RNA was extracted from cells using TrizolH reagent (Invitrogen) according to manufacturer’s recommendations. 1? mg RNA was used for first-strand cDNA synthesis with the SuperScript II system (Invitrogen) and oligo-dT primers. qPCR analyses were performed on the LightCyclerTCTP in Prostate CancerFigure 3. Downregulation of TCTP expression leads to increased apoptosis in LNCaP cells. LNCaP cells were cultured on coverslips and transfected with siRNA against TCTP or Luciferase (Luc) for 72 h. Apoptosis was induced by 100 nM TG for 36 h during siRNA treatment. After treatment and fixation, apoptotic cell death was detected using the TUNEL assay, cell nuclei were stained with DAPI and cells were visualized using Axioplan imaging microscope. A. Representative pictures of LNCaP cells transfected with either TCTP- or Luc-siRNA for 72 h and treated with DSMO or TG for 36 h during transfection. TUNEL positive cells appear as red spots. Arrows indicate apoptotic cells. B. Quantification of apoptosis incidence. The data show the percentage of nonviable cells after 36 h treatment with TG in cells transfected with TCTP or Luc siRNA. Columns represent the mean of three independent experiments performed in triplicate and bars.