Journal: British Journal of Nutrition

Article Title: Maslinic acid inhibits the metastatic capacity of DU145 human prostate cancer cells: possible mediation via hypoxia-inducible factor-1α signalling

doi: 10.1017/s0007114512000967

Figure Lengend Snippet: Fig. 1. Maslinic acid inhibits the migration and invasion of DU145 and TRAMP-C2 cells. Cells were serum-deprived in Dulbecco’s modified Eagle’s medium (DMEM)/nutrient mixture Ham’s F12 (F12) containing 1 % charcoal-stripped fetal bovine serum (FBS) for 24 h. (A, B) DU145 cells and (C) TRAM-C2 cells were plated on 6·5 mm transwell filters precoated with (A, C) type IV collagen or (B) Matrigel at 25 000 cells/well and treated with 0–25 mM-maslinic acid. The lower compartment was filled with DMEM/F12 medium supplemented with 1 % charcoal-stripped FBS and 0·1 % bovine serum albumin with or without epidermal growth factor (EGF). Cells were incubated for (A) 4, (B) 12 or (C) 1 h. (A, C) Migrating and (B) invading cells were quantified. Values are means from three independent experiments, with standard errors represented by vertical bars. a,b,c,d,e Mean values with unlike letters were significantly different (P,0·05).

Article Snippet: DU145 and TRAMP-C2 mouse prostate cancer cells were obtained from the American Type Culture Collection.

Techniques: Migration, Incubation

Journal: British Journal of Nutrition

Article Title: Maslinic acid inhibits the metastatic capacity of DU145 human prostate cancer cells: possible mediation via hypoxia-inducible factor-1α signalling

doi: 10.1017/s0007114512000967

Figure Lengend Snippet: Fig. 2. Effect of maslinic acid on the secretion of matrix metalloproteinase (MMP) and tissue inhibitors of metalloproteinases (TIMP) in DU145 cells. DU145 cells were plated in 100 mm dishes at 2 £ 106 cells/dish in Dulbecco’s modified Eagle’s medium (DMEM)/nutrient mixture Ham’s F12 (F12) supplemented with 10 % fetal bovine serum (FBS). At 1 d later, the monolayers were serum-deprived in DMEM/F12 medium containing 1 % charcoal-stripped FBS for 24 h. Cells were incu- bated with 0–25 mM-maslinic acid in serum-free medium with or without epidermal growth factor (EGF) for 18 h. The 18 h conditioned media were concentrated for (A) gelatin zymography and (C) Western blotting. In the first lane of (A), serum-free HT-1080 cell-conditioned medium was loaded. The volumes of media loaded onto the gel were adjusted for equivalent protein concentrations. Photographs of chemiluminescent detection of Coomassie blue-stained (A) gels and (C) blots, which are representative of three independent experiments. The relative abundance of each band was quantified by densitometric scanning of the exposed film. The adjusted means of each band with their standard errors are shown above each blot. (B) Serum-deprived cells were incubated for 12 h with maslinic acid and/or EGF. Total RNA was isolated and reverse-transcribed, and real-time PCR was conducted. MMP-9 mRNA expression was normalised to that of glyceralde- hyde-3-phosphate dehydrogenase (GAPDH). Values are means, with standard errors represented by vertical bars (n 3). a,b,c,d,e Mean values with unlike letters were significantly different (P , 0·05). ND, not determined; Mr, molecular weight.

Article Snippet: DU145 and TRAMP-C2 mouse prostate cancer cells were obtained from the American Type Culture Collection.

Techniques: Zymography, Western Blot, Staining, Incubation, Isolation, Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Molecular Weight

Journal: British Journal of Nutrition

Article Title: Maslinic acid inhibits the metastatic capacity of DU145 human prostate cancer cells: possible mediation via hypoxia-inducible factor-1α signalling

doi: 10.1017/s0007114512000967

Figure Lengend Snippet: Fig. 3. Maslinic acid inhibits urokinase-type plasminogen activator (uPA) activity in DU145 cells. DU145 cells were plated, serum-deprived and treated with masli- nic acid as described in Fig. 2, and 18 h conditioned media were collected. (A) To determine uPA secretion and uPA receptor (uPAR) expression, volumes of media and total cell lysates, respectively, loaded onto the gel were adjusted for equivalent protein concentrations. Photographs of chemiluminescent detection of the Western blots, which are representative of three independent experiments, are shown. The relative abundance of each band was quantified by densitometric scanning of the exposed film. uPAR and plasminogen activator inhibitor 1 (PAI-1) levels were normalised to those of b-actin. The adjusted means of each band with their standard errors are shown above each blot. (B) uPA activity was with unlike letters determined using unconcentrated conditioned media. (C) Serum- deprived cells were incubated with maslinic acid and/or epidermal growth factor (EGF) for 12 h. Total RNA was isolated and reverse-transcribed, and real-time PCR was conducted. The levels of uPA mRNA were normalised to those of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Values are means, with stan- dard errors represented by vertical bars (n 3). a,b,c,d,e Mean values were significantly different (P,0·05). ND, not determined; Mr, molecular weight.

Article Snippet: DU145 and TRAMP-C2 mouse prostate cancer cells were obtained from the American Type Culture Collection.

Techniques: Activity Assay, Expressing, Western Blot, Incubation, Isolation, Reverse Transcription, Real-time Polymerase Chain Reaction, Molecular Weight

Journal: British Journal of Nutrition

Article Title: Maslinic acid inhibits the metastatic capacity of DU145 human prostate cancer cells: possible mediation via hypoxia-inducible factor-1α signalling

doi: 10.1017/s0007114512000967

Figure Lengend Snippet: Fig. 4. Maslinic acid inhibits adhesion of DU145 cells. (A) Serum-deprived DU145 cells were plated in CytoMatrixe human collagen I cell adhesion strips (Chemi- con International) and were incubated for 45 min in Dulbecco’s modified Eagle’s medium/nutrient mixture Ham’s F12 containing 1 % charcoal-stripped fetal bovine serum with 0–25 mM-maslinic acid with or without epidermal growth factor (EGF). Cells were stained with 0·2 % crystal violet, and the cell-bound stains were quan- tified colorimetrically. Values are means, with standard errors represented by vertical bars (n 4). (B) Serum-deprived DU145 cells were treated with maslinic acid in serum-deprived media. Total cell lysates were subjected to immunoblotting with antibodies raised against intercellular adhesion molecule (ICAM), vascular cell adhesion molecule (VCAM) or E-cadherin. Photographs of chemiluminescent detection of the blots, which are representative of three independent experiments, are shown. The relative abundance of each band was estimated by densitometric scanning of the exposed films, and the expression levels were normalised to those of b-actin. The adjusted means (n 3) of each band with their standard errors are shown above each blot. a,b,c,d Mean values with unlike letters were signifi- cantly different (P,0·05). Mr, molecular weight.

Article Snippet: DU145 and TRAMP-C2 mouse prostate cancer cells were obtained from the American Type Culture Collection.

Techniques: Incubation, Staining, Western Blot, Expressing, Molecular Weight

Journal: British Journal of Nutrition

Article Title: Maslinic acid inhibits the metastatic capacity of DU145 human prostate cancer cells: possible mediation via hypoxia-inducible factor-1α signalling

doi: 10.1017/s0007114512000967

Figure Lengend Snippet: Fig. 6. Maslinic acid reduces hypoxia-inducible factor (HIF)-1a protein levels in DU145 cells. (A, B) Serum-deprived cells were incubated with 0–25 mM-maslinic acid in serum-deprived media with or without epidermal growth factor (EGF) for 6 h under normoxic or hypoxic conditions. (E) Serum-deprived cells were stimulated with EGF for 6 h and then treated with 100 mM-cycloheximide (CHX) with or without 10 mM-maslinic acid for the indicated periods. Total cell lysates (A, E) and the nuclear fractions (B) were subjected to Western blotting with an HIF-1a antibody. The relative abundance of each band was quantified by densitometric scanning of the exposed films, and the expression levels were normalised to those of b-actin. The adjusted means of each band with their standard errors are shown above each blot. (C) Cells were co-transfected with a human hypoxia-responsive element (HRE) reporter gene construct and a pRL-TK control vector. The transfected cells were plated in twenty-four-well plates at 25 000 cells/well. After serum deprivation, cells were incubated with the indicated concentrations of maslinic acid in serum- deprived media with or without EGF for 6 h. Cell lysates were prepared to measure luciferase and Renilla activities. Luciferase activity was normalised to Renilla activity. (D) Serum-deprived cells were incubated for 12 h with maslinic acid and/or EGF. Total RNA was isolated and reverse-transcribed, and real-time PCR was conducted. HIF-1a mRNA expression was normalised to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Values are means (n 3), with standard errors represented by vertical bars. a,b,c,d Mean values with unlike letters were significantly different (P , 0·05). ND, not determined; Mr, molecular weight.

Article Snippet: DU145 and TRAMP-C2 mouse prostate cancer cells were obtained from the American Type Culture Collection.

Techniques: Incubation, Western Blot, Expressing, Transfection, Construct, Control, Plasmid Preparation, Luciferase, Activity Assay, Isolation, Reverse Transcription, Real-time Polymerase Chain Reaction, Molecular Weight

Journal: British Journal of Nutrition

Article Title: Maslinic acid inhibits the metastatic capacity of DU145 human prostate cancer cells: possible mediation via hypoxia-inducible factor-1α signalling

doi: 10.1017/s0007114512000967

Figure Lengend Snippet: Fig. 7. Maslinic acid reduces the activation of Akt and extracellular signal-related kinase (ERK) in DU145 cells. (A) Serum-deprived cells were incubated with 0– 25 mM-maslinic acid for 6 h, and cell lysates were prepared with or without a 15 min epidermal growth factor (EGF) stimulation. Serum-deprived cells were incu- bated with (B) 0–20 mM-LY294002 or (C) 0–40 mM-PD98059 for 6 h and lysed without stimulation or after 15 min of EGF stimulation to determine phospho (p)-Akt or p-ERK1/2 levels. To identify hypoxia-inducible factor-1a (HIF-1a), serum-deprived cells were incubated with (B) 0–20 mM-LY294002 or (C) 0–40 mM-PD98059 in the absence or presence of EGF for 6 h. Total cell lysates were subjected to Western blotting. Photographs of chemiluminescent detection of the blots, which are representative of three independent experiments, are shown. The relative abundance of each band was estimated by densitometric scanning of the exposed films, and the expression levels were normalised to those of b-actin. To determine vascular endothelial growth factor (VEGF) concentrations, serum-deprived cells were incubated with (B) 0–20 mM-LY294002 or (C) 0–40 mM-PD98059 in serum-free media with or without 10 mg/l EGF for 18 h. The 18 h conditioned media were concentrated and subjected to Western blotting with VEGF antibody. The volumes of media loaded onto the gel were adjusted for equivalent protein concen- trations. Photographs of chemiluminescent detection of the blots, which are representative of three independent experiments, are shown. The relative abundance of each band was quantified by densitometric scanning of the exposed films. The adjusted means (n 3) of each band with their standard errors are shown above each blot. a,b,c,d Mean values with unlike letters were significantly different (P , 0·05). Mr, molecular weight.

Article Snippet: DU145 and TRAMP-C2 mouse prostate cancer cells were obtained from the American Type Culture Collection.

Techniques: Activation Assay, Incubation, Western Blot, Expressing, Molecular Weight

Journal: Pharmacological research

Article Title: A selective alpha1D-adrenoreceptor antagonist inhibits human prostate cancer cell proliferation and motility "in vitro".

doi: 10.1016/j.phrs.2015.11.017

Figure Lengend Snippet: Figure 4: Cell cycle analysis and p27 and p21 levels in PC3 and DU145 cells exposed to A175.

Article Snippet: Human androgen independent prostate cancer PC3 and DU145 cell lines and the androgendependent LNCaP cell line were originally obtained from American Type Culture Collection (ATCC, Rockville, MD, USA); CWR22Rv1 (Rv1) cells were originally obtained from LeibnitzInstitut DSMZ (Braunschweig, Germany).

Techniques: Cell Cycle Assay

Journal: Pharmacological research

Article Title: A selective alpha1D-adrenoreceptor antagonist inhibits human prostate cancer cell proliferation and motility "in vitro".

doi: 10.1016/j.phrs.2015.11.017

Figure Lengend Snippet: Figure 5: Effect of A175 on PC3 and DU145 cell motility.

Article Snippet: Human androgen independent prostate cancer PC3 and DU145 cell lines and the androgendependent LNCaP cell line were originally obtained from American Type Culture Collection (ATCC, Rockville, MD, USA); CWR22Rv1 (Rv1) cells were originally obtained from LeibnitzInstitut DSMZ (Braunschweig, Germany).

Techniques:

Journal: Oncotarget

Article Title: DNA mismatch repair gene MLH1 induces apoptosis in prostate cancer cells

doi:

Figure Lengend Snippet: Prostatic cell lines were grown in culture dishes for two days. (A) Representative immunoblot displaying MLH1 protein expression in DU145 and normal prostate epithelial PWR-1E and RWPE-1 cells. GAPDH was used as loading control. (B) Relative mRNA expression levels of MLH1 in cell lines as determined by real-time PCR. Expression levels are normalized to PWR-1E. Data are presented as mean±SEM of three experiments. Asterisks denote differences between the compared values: *P<0.05, **P<0.01.

Article Snippet: DU145 cells were transfected with pCMV6-ENTRY vector expressing the C-terminally Myc and Flag-tagged human MLH1 cDNA as well as empty pCMV6-ENTRY vector as a control (OriGene Techologies, Rockville, MD) using X-treme Gene HD transfection reagent (Roche Diagnostics, Indianapolis, IN) according to the manufacturer's protocol.

Techniques: Western Blot, Expressing, Real-time Polymerase Chain Reaction

Journal: Oncotarget

Article Title: DNA mismatch repair gene MLH1 induces apoptosis in prostate cancer cells

doi:

Figure Lengend Snippet: (A) Ectopic expression of MLH1. DU145 cells stably transfected with either MLH1 or empty vector (pCMV) along with mock (parental DU145 cells treated with transfection reagent alone) were grown for 48 hours and underwent Western analyses. GAPDH was used as loading control. (B) Cell proliferation as analyzed by the MTS cell proliferation assay 48 hours after plating cells. Results are expressed as % and normalized to pCMV control. (C) Cell migration as measured by wound healing assay. A wound was formed by scraping culture dishes using a pipet tip and closure measured after 24 hours. Left: Representative images of wound healing assay are shown. Right: Migration expressed as % closure of wound. (D) Cell invasiveness as measured using Matrigel. Cells were placed onto transwell membrane and allowed to invade for 24 hours. Left: Representative images of invading cells are shown. Right: Cell invasiveness as measured by absorbance (Abs) at 560 nm. Data are presented as mean±SEM of at least three experiments; **P<0.01 MLH1 versus pCMV.

Article Snippet: DU145 cells were transfected with pCMV6-ENTRY vector expressing the C-terminally Myc and Flag-tagged human MLH1 cDNA as well as empty pCMV6-ENTRY vector as a control (OriGene Techologies, Rockville, MD) using X-treme Gene HD transfection reagent (Roche Diagnostics, Indianapolis, IN) according to the manufacturer's protocol.

Techniques: Expressing, Stable Transfection, Transfection, Plasmid Preparation, Western Blot, Proliferation Assay, Migration, Wound Healing Assay

Journal: Oncotarget

Article Title: DNA mismatch repair gene MLH1 induces apoptosis in prostate cancer cells

doi:

Figure Lengend Snippet: Athymic nude mice were injected subcutaneously with stable MLH1 or pCMV-transfected DU145 cells and growth monitored over time. Left: Representative image of tumors in mice five weeks after injection of cells. Right: Growth of tumor size plotted over time. Data are presented as mean±SEM of five mice per group; *P<0.05 MLH1 versus pCMV for each time point.

Article Snippet: DU145 cells were transfected with pCMV6-ENTRY vector expressing the C-terminally Myc and Flag-tagged human MLH1 cDNA as well as empty pCMV6-ENTRY vector as a control (OriGene Techologies, Rockville, MD) using X-treme Gene HD transfection reagent (Roche Diagnostics, Indianapolis, IN) according to the manufacturer's protocol.

Techniques: Injection, Transfection

Journal: Oncotarget

Article Title: DNA mismatch repair gene MLH1 induces apoptosis in prostate cancer cells

doi:

Figure Lengend Snippet: Stable MLH1-expressing and vector control DU145 cells were grown over time. (A) Apoptosis as measured by flow cytometric analyses. Representative biparametric histogram showing cell population in early (bottom right quadrant) and late (top right quadrant) apoptotic, and viable (bottom left quadrant) states. Left: pCMV control, Middle: MLH1-expressing, Right: Total apoptosis %, Upper: 48 hours of growth, Lower: 72 hours of growth. Bar graph of total apoptosis % is presented as mean±SEM of three experiments, *P<0.05 MLH1 versus pCMV. (B) Protein levels of MLH1, cleaved PARP (cl-PARP), c-Abl, and p-c-Abl were determined by Western blot analyses after growing pCMV and MLH1 cells for 48 hours. GAPDH was used as loading control.

Article Snippet: DU145 cells were transfected with pCMV6-ENTRY vector expressing the C-terminally Myc and Flag-tagged human MLH1 cDNA as well as empty pCMV6-ENTRY vector as a control (OriGene Techologies, Rockville, MD) using X-treme Gene HD transfection reagent (Roche Diagnostics, Indianapolis, IN) according to the manufacturer's protocol.

Techniques: Expressing, Plasmid Preparation, Western Blot

Journal: Oncotarget

Article Title: DNA mismatch repair gene MLH1 induces apoptosis in prostate cancer cells

doi:

Figure Lengend Snippet: Three MLH1 siRNAs (siMLH1s) were transfected individually along with a non-specific siRNA control (siControl) into stable MLH1-expressing DU145 cells for 48 hours. pCMV control cells treated with Lipofectamine are also included. (A) Protein levels of MLH1, c-Abl, and p-c-Abl in cells as determined by Western blot analyses. GAPDH was used as loading control. (B) Total apoptotic cells were analyzed by flow cytometry. Data are expressed as mean±SEM of three experiments; *P<0.05 **P<0.01 siMLH1s versus siControl.

Article Snippet: DU145 cells were transfected with pCMV6-ENTRY vector expressing the C-terminally Myc and Flag-tagged human MLH1 cDNA as well as empty pCMV6-ENTRY vector as a control (OriGene Techologies, Rockville, MD) using X-treme Gene HD transfection reagent (Roche Diagnostics, Indianapolis, IN) according to the manufacturer's protocol.

Techniques: Transfection, Expressing, Western Blot, Flow Cytometry

Journal: Oncotarget

Article Title: DNA mismatch repair gene MLH1 induces apoptosis in prostate cancer cells

doi:

Figure Lengend Snippet: MLH1-expressing and pCMV control DU145 transfectants were treated with either DMSO or STI571 and maintained for 48 hours. Total apoptotic cells were then analyzed by flow cytometry. Data are expressed as mean±SEM of three experiments; **P<0.01 MLH1+/STI571+ versus MLH1+/STI571-.

Article Snippet: DU145 cells were transfected with pCMV6-ENTRY vector expressing the C-terminally Myc and Flag-tagged human MLH1 cDNA as well as empty pCMV6-ENTRY vector as a control (OriGene Techologies, Rockville, MD) using X-treme Gene HD transfection reagent (Roche Diagnostics, Indianapolis, IN) according to the manufacturer's protocol.

Techniques: Expressing, Flow Cytometry

Journal: PLoS ONE

Article Title: Withaferin A Induces Cell Death Selectively in Androgen-Independent Prostate Cancer Cells but Not in Normal Fibroblast Cells

doi: 10.1371/journal.pone.0134137

Figure Lengend Snippet: (A, B) Western blot analysis to detect c-Fos (FosB) in TIG-1, LNCaP, DU-145, and PC-3 cells at 4, 8, and 24 h after treatment with 4 μM (A) or 2 μM (B) WA. NT, non-treated. (C) Western blot analysis to detect c-Fos, PARP, FLIP and GAPDH in PC-3 cells at 12 h after 4 μM WA treatment in the presence (+) or absence (-) of three different siRNAs (X–Z) from OriGene. (D) Viability of PC-3 cells after siFos treatment. Data are represented as means ± SEM of three independent experiments; pink arrows indicate a statistically significant reduction in cell number following siFos treatment (**, P < 0.01). (E) Population of apoptotic, necrotic, and live cells distinctly stained with Annexin V–EnzoGold, 7-AAD-Red, and GFP. Data are represented as means ± SEM of three independent experiments; red, blue, and green arrows indicate statistically significant changes (**, P < 0.01). (F) Western blot analysis of c-Fos, PARP, FLIP, and GAPDH in DU-145 at 12 h after 4 μM WA treatment in the presence (+) or absence (-) of siRNAs; siFos or siGL2 (siControl). (G) Viability of DU-145 cells after exogenous overexpression of pcDNA3-FLIP or vector alone in the presence of DMSO (solvent) or 4 μM WA.

Article Snippet: Human cells were acquired from the indicated suppliers: PC-3 and DU-145 (RIKEN Bioresource Center), TIG-1 (Japanese Cancer Research Resources Bank), KD (Japanese Collection of Research Bioresources Cell Bank) and LNCaP (ATCC).

Techniques: Western Blot, Staining, Over Expression, Plasmid Preparation

Journal: PLoS ONE

Article Title: Withaferin A Induces Cell Death Selectively in Androgen-Independent Prostate Cancer Cells but Not in Normal Fibroblast Cells

doi: 10.1371/journal.pone.0134137

Figure Lengend Snippet: (A) Western blot analysis to detect HSPA6, HSP40, HSPA70, EGR-1, PAR-4, and GAPDH (loading control) in TIG-1, LNCaP, DU-145, and PC-3 cells at 0, 4, 8 and 24 h after 4 μM WA treatment. Arrow indicates the band for bona fide EGR-1. (B) Influence of siHSPA6 expression on PC-3 cell growth. (i) Schematic for this experiment. (ii) Western blot analysis to confirm the knockdown of HSPA6 protein by siHSPA6, relative to cells treated with siGL2 (negative control). (iii) Influence of siHSPA6 and siGL2 on PC-3 cell growth, with or without WA treatment. Arrow highlights the reduction in PC-3 cell growth. (C) Influence of siHSF-1 expression on PC-3 cell growth. (i) Schematics for this experiment. (ii) Western blot analysis to confirm the knockdown of HSP protein by siHSF-1, relative to cells treated with siGL2 (negative control), and to determine whether HSF-1 knockdown affected HSP protein levels by detection of HSPA6, HSP40, HSPA70, and GAPDH in PC-3 cells. (iii) Influence of siHSF-1 and siGL2 on PC-3 cell growth, with or without WA treatment. Arrow highlights the reduction in PC-3 cell growth. Data are represented as means ± SEM of three independent experiments; pink, green, and blue arrows indicate significant decreases in cell viabilities (*, P < 0.05; **, P < 0.01).

Article Snippet: Human cells were acquired from the indicated suppliers: PC-3 and DU-145 (RIKEN Bioresource Center), TIG-1 (Japanese Cancer Research Resources Bank), KD (Japanese Collection of Research Bioresources Cell Bank) and LNCaP (ATCC).

Techniques: Western Blot, Expressing, Negative Control

Journal: PLoS ONE

Article Title: Withaferin A Induces Cell Death Selectively in Androgen-Independent Prostate Cancer Cells but Not in Normal Fibroblast Cells

doi: 10.1371/journal.pone.0134137

Figure Lengend Snippet: (A) Western blot analysis to detect IRE-1α, PERK, pS51-eIF2α, CHOP, caspase 3, PARP, BiP, and GAPDH (loading control) in TIG-1, LNCaP, DU-145, and PC-3 cells at 4, 8, and 24 h after 4 μM WA treatment. NT: non-treated. (B) Schematic representation of the analyzed proteins in the apoptotic pathway induced by ER stress. (C) Typical fluorescence images of TIG-1 (i), LNCaP (ii), PC-3 (iii), and DU-145 (iv) that were treated with 4 μM WA for 24 h and subsequently treated with ROS detection reagents using the Image-iT LIVE Green ROS Detection Kit. Merged images are shown of cytoplasmic ROS signals (green) and nuclear DNA signals stained with Hoechst33342 (blue). Green bar, 100 μm. Black bar, 10 μm.

Article Snippet: Human cells were acquired from the indicated suppliers: PC-3 and DU-145 (RIKEN Bioresource Center), TIG-1 (Japanese Cancer Research Resources Bank), KD (Japanese Collection of Research Bioresources Cell Bank) and LNCaP (ATCC).

Techniques: Western Blot, Fluorescence, Staining

Journal: PLoS ONE

Article Title: Withaferin A Induces Cell Death Selectively in Androgen-Independent Prostate Cancer Cells but Not in Normal Fibroblast Cells

doi: 10.1371/journal.pone.0134137

Figure Lengend Snippet: (A) Western blot analysis to detect vimentin and actin in TIG-1, LNCaP, PC-3, and DU-145 at 4, 8, and 24 h after treatment with 2 μM or 4 μM WA. NT: non-treated. (B) Typical images of TIG-1 (i) and PC-3 (ii) stained with anti-vimentin antibody, phalloidin (for F-actin), and Hoechst 33342 (for DNA) at 2, 4, and 6 (h) after WA treatment. NT: non-treated. Pink and yellow arrows indicated colocalized vimentin and F-actin aggregates. Merged images are shown in the bottom row. Bar, 10 μm. (C) Percentage of all observed cells containing dots of aggregated or non-aggregated vimentin are shown for TIG-1 (i) and PC-3 (ii). Cells harboring over 10 vimentin aggregates were scored as “aggregated”, whereas cells with non-aggregated vimentin were scored as “non-aggregated”. Data are represented as means ± SEM of three independent experiments; asterisks indicate statistically significant changes in cell viability (**, P < 0.01; ***, P < 0.001). (D) Jasplakinoloide (Jsp) did not influence PC-3 cell viability. (i) Schematic of the experimental design. (ii) Bar graphs showing cell viability (%) in the presence (+) or absence (-) of 4 μM WA or Jasp 8 h after the addition of WA. NT: non-treated. Bar graphs represent means ± SEM for three independent experiments. Pink, blue, and green arrows indicate statistically significant reductions in cell viability (*, P < 0.05; **, P < 0.01).

Article Snippet: Human cells were acquired from the indicated suppliers: PC-3 and DU-145 (RIKEN Bioresource Center), TIG-1 (Japanese Cancer Research Resources Bank), KD (Japanese Collection of Research Bioresources Cell Bank) and LNCaP (ATCC).

Techniques: Western Blot, Staining

Journal: PLoS ONE

Article Title: Withaferin A Induces Cell Death Selectively in Androgen-Independent Prostate Cancer Cells but Not in Normal Fibroblast Cells

doi: 10.1371/journal.pone.0134137

Figure Lengend Snippet: (A) Frequency of spheres larger than 100 μm (i) and typical images (ii) after incubation of TIG-1, LNCaP, PC-3, DU-145, and SAS cells in sphere-formation medium for 10 days. (B) Viability of SAS cells after WA treatment. Bars represent means ± SEM of three independent experiments. Cell viability was measured at 4, 8, and 24 h after treatment with 2 μM (blue bars) or 4 μM (red bars) WA. NT, non-treated. Data are represented as means ± SEM of three independent experiments; red arrows indicate statistically significant reductions in cell viability (**, P < 0.01). (C) Western blot analysis to detect c-Fos, HSPA6, HSP40, HSP70, PARP, and GAPDH (loading control) in SAS cells at 4, 8, and 24 h after treatment with 2 μM or 4 μM WA. NT: non-treated. (D) Comparison of sensitivity to cisplatin between parental cells and spheres of PC-3 (i and ii) and SAS (iii and iv) after 48 h of incubation following treatment with indicated cisplatin concentrations.

Article Snippet: Human cells were acquired from the indicated suppliers: PC-3 and DU-145 (RIKEN Bioresource Center), TIG-1 (Japanese Cancer Research Resources Bank), KD (Japanese Collection of Research Bioresources Cell Bank) and LNCaP (ATCC).

Techniques: Incubation, Western Blot