Journal: Cancer Cell International

Article Title: Decreased intracellular zinc in human tumorigenic prostate epithelial cells: a possible role in prostate cancer progression

doi: 10.1186/1475-2867-6-10

Figure Lengend Snippet: mRNA expression of zinc uptake transporters in RWPE1 and RWPE2. RWPE1 and RWPE2 were grown to ~50% confluence and treated with 0 or 75 μM ZnSO 4 at 37°C for 48 hours. Cells were harvested and total RNA was isolated. Gene expression of ZIP1 and ZIP3 were measured by quantitative RT-PCR with TaqMan probes and normalized to the β-actin expression. The gene expression levels are presented as copy numbers/10 4 β-actin transcripts. Values are the means ± SE, n = 6 (two independent experiments).

Article Snippet: The ZIP1-Myc expressing cell line and vector control cell line were generated by transfecting pcDNA3.1/ZIP1-Myc or pcDNA3.1/Myc-His (Invitrogen) into RWPE2 using LipofectAMINE plus kit (Invitrogen).

Techniques: Expressing, Isolation, Quantitative RT-PCR

Journal: Cancer Cell International

Article Title: Decreased intracellular zinc in human tumorigenic prostate epithelial cells: a possible role in prostate cancer progression

doi: 10.1186/1475-2867-6-10

Figure Lengend Snippet: Subcellular localization of ZIP1 and ZIP3 in RWPE1 and RWPE2. a . Localization of ZIP1 and ZIP3. RWPE1 and RWPE2 were seeded at ~50% confluence in a 4-well slide chamber and cultured at 37°C for 48 hours. Cells were then treated with 0 or 50 μM ZnSO 4 at 37°C for 2 hours before immunofluorescent staining. The endogenous ZIP1 and ZIP3 proteins in RWPE1 and RWPE2 were detected by a chicken anti-ZIP1 antibody and a rabbit anti-ZIP3 antibody, respectively. An Alexa 488-conjugated goat anti-chicken (ZIP1) or anti-rabbit (ZIP3) antibody were used as secondary antibodies for the photomicrographs. Arrows indicate the plasma membrane localization of ZIP1. b . Localization of ZIP3 and LAMP1 in RWPE2. RWPE2 were seeded at ~50% confluence in a 4-well slide chamber and cultured at 37°C for 48 hours. Cells were fixed, permeablized, and stained as described in the Methods. The green (ZIP3, panel A) and red (LAMP1, panel B) florescent images were merged to indicate the area of overlap (panel C), which is shown in yellow .

Article Snippet: The ZIP1-Myc expressing cell line and vector control cell line were generated by transfecting pcDNA3.1/ZIP1-Myc or pcDNA3.1/Myc-His (Invitrogen) into RWPE2 using LipofectAMINE plus kit (Invitrogen).

Techniques: Cell Culture, Staining

Journal: Cancer Cell International

Article Title: Decreased intracellular zinc in human tumorigenic prostate epithelial cells: a possible role in prostate cancer progression

doi: 10.1186/1475-2867-6-10

Figure Lengend Snippet: Expression of the ZIP1 protein in RWPE1 and RWPE2. a . Detection of the ZIP1 protein. Western blot containing 50 μg of protein extracts isolated from RWPE1 and RWPE2 cells were probed with a chicken anti-ZIP1 antibody followed by a peroxidase-conjugated secondary anti-chicken antibody. ZIP1 was visualized using an ECL kit (Amersham Biosciences). The detection of GRP78 on the same western blot was served as loading control. The protein markers (Bio-Rad) are shown. b . Quantitation of the expression level of the ZIP1 protein. The ZIP1 and GRP78 signals from the western blot (Fig. 5a) were quantified by an Alpha Innotech Gel Documentation System (Alpha Innotech). The expression of ZIP1 was normalized by the expression of GRP78. c . Regulation of the ZIP1 protein expression by zinc. RWPE1 cells were grown for 24 hours and treated with 0, 25, or 50 μM ZnSO 4 for 24 hours. Cell lysate was prepared and analyzed by western blot assay. The ZIP1 and GRP78 proteins were detected as described as above.

Article Snippet: The ZIP1-Myc expressing cell line and vector control cell line were generated by transfecting pcDNA3.1/ZIP1-Myc or pcDNA3.1/Myc-His (Invitrogen) into RWPE2 using LipofectAMINE plus kit (Invitrogen).

Techniques: Expressing, Western Blot, Isolation, Quantitation Assay

Journal: Cancer Cell International

Article Title: Decreased intracellular zinc in human tumorigenic prostate epithelial cells: a possible role in prostate cancer progression

doi: 10.1186/1475-2867-6-10

Figure Lengend Snippet: Over-expression of ZIP1-Myc in RWPE2. RWPE2 was stably transfected with an empty vector or a ZIP1-myc expressing plasmid. a . Expression of the ZIP1-Myc mRNA in two stably transfected RWPE2 cell lines. Vector or ZIP-Myc transfected (Clone A and Clone B) RWPE2 were seeded at ~50% confluence and 0 or 25 μM ZnSO 4 was added into the medium after 24 hours incubation. Cells were then incubated at 37°C for another 24 hours and total RNA was isolated. The endogenous ZIP1 as well as ZIP1-Myc mRNA were measured by quantitative RT-PCR with a TaqMan probe specific for ZIP1. The expression of ZIP1 mRNA was normalized to the β-actin mRNA expression. Values are the means ± SD of three experiments. b . Expression and localization of ZIP1-Myc in stably transfected RWPE2. Vector and ZIP1-Myc transfected RWPE2 were seeded at ~25% confluence and cultured at 37°C for 48 hours. ZIP1-Myc was detected by a mouse anti-Myc antibody (panel B). Arrow indicates the plasma membrane localization of ZIP1-Myc. The Myc staining of RWPE2 stably transfected with the vector control is shown in panel A. c . Expression of ZNT1 mRNA in stably transfected RWPE2. Vector or ZIP1-Myc expressing (Clone A and Clone B) RWPE2 were seeded at ~50% confluence and 0 or 25 μM ZnSO 4 was added into the medium after 24 hours incubation. Cells were then incubated at 37°C for another 24 hours and total RNA was isolated. The endogenous ZNT1 mRNA was measured by quantitative RT-PCR with a TaqMan probe specific for ZNT1. The expression of ZNT1 was normalized to the β-actin expression. Values are the means ± SD of three experiments.

Article Snippet: The ZIP1-Myc expressing cell line and vector control cell line were generated by transfecting pcDNA3.1/ZIP1-Myc or pcDNA3.1/Myc-His (Invitrogen) into RWPE2 using LipofectAMINE plus kit (Invitrogen).

Techniques: Over Expression, Stable Transfection, Transfection, Plasmid Preparation, Expressing, Incubation, Isolation, Quantitative RT-PCR, Cell Culture, Staining

Journal: Cancer Cell International

Article Title: Decreased intracellular zinc in human tumorigenic prostate epithelial cells: a possible role in prostate cancer progression

doi: 10.1186/1475-2867-6-10

Figure Lengend Snippet: Effect of over-expression of ZIP1-Myc on RWPE2 cell growth. Vector or ZIP1-Myc expressing (Clone A and Clone B) RWPE2 were plated at a density of 20,000 per well in 12-well plates for 24 hours and then 0 or 25 μM ZnSO 4 was added into the medium. Medium was changed at days 3, 6 and 9 after plating. Cells were harvested and cell numbers were determined at days 6, 9, and 12 after plating. Values are the means ± SD of three experiments.

Article Snippet: The ZIP1-Myc expressing cell line and vector control cell line were generated by transfecting pcDNA3.1/ZIP1-Myc or pcDNA3.1/Myc-His (Invitrogen) into RWPE2 using LipofectAMINE plus kit (Invitrogen).

Techniques: Over Expression, Plasmid Preparation, Expressing

Journal: Cancer Cell International

Article Title: Decreased intracellular zinc in human tumorigenic prostate epithelial cells: a possible role in prostate cancer progression

doi: 10.1186/1475-2867-6-10

Figure Lengend Snippet: Effect of over-expression of ZIP1-Myc on RWPE2 cell cycle progression and apoptosis. a . Effect of over-expression of ZIP1 on cell cycle. Vector or ZIP1-Myc expressing RWPE2 cells were plated and grown for 24 hours. Cells were harvested and counted. 2 × 10 6 cells were ethanol fixed and processed for DNA staining with propidium Iodine. Cells were run on a FACS SCAN (BD Dickenson) with a 488 nm argon laser light source. 10,000 events were counted per sample. Values are the means ± SD of one representative experiment of two with similar results. b . Effect of over-expression of ZIP1 on apoptosis. 2 × 10 6 vector or ZIP1-Myc expressing RWPE2 cells were plated in 100 mm plates and grown for 24 hours. Cells were treated with TRAIL (10 ng/ml) at 37°C for 6 hours. Cells were harvested by trypsinization. Caspase 3 activity was determined according to the manufacture's instructions (Clontech ApoAlert Caspase Colorimetric Assay Kit). Values are means ± SD of one representative experiment of two with similar results.

Article Snippet: The ZIP1-Myc expressing cell line and vector control cell line were generated by transfecting pcDNA3.1/ZIP1-Myc or pcDNA3.1/Myc-His (Invitrogen) into RWPE2 using LipofectAMINE plus kit (Invitrogen).

Techniques: Over Expression, Plasmid Preparation, Expressing, Staining, Activity Assay, Colorimetric Assay

Journal: Journal of Inflammation (London, England)

Article Title: Effect of Serenoa repens (Permixon®) on the expression of inflammation-related genes: analysis in primary cell cultures of human prostate carcinoma

doi: 10.1186/1476-9255-10-11

Figure Lengend Snippet: Cell counting by trypsan blue in LNCaP and PC3 cell lines. Results are presented at 24, 48 and 72 hours of incubation, either in untreated or in Permixon® treated (44 and 88 μg/ml) conditions. Cells were seeded at different concentration, lower for longer incubation time in order to avoid overgrowth. The same amount of cells was used to start each incubation time, control and treated cells. a) LNCaP. b) PC3. * = p < 0.05.

Article Snippet: LNCaP and PC3 cell lines were obtained from Interlab Cell Line Collection (ICLC) (Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy).

Techniques: Cell Counting, Incubation, Concentration Assay, Control

Journal: Journal of Inflammation (London, England)

Article Title: Effect of Serenoa repens (Permixon®) on the expression of inflammation-related genes: analysis in primary cell cultures of human prostate carcinoma

doi: 10.1186/1476-9255-10-11

Figure Lengend Snippet: Cytitoxicity assay in LNCaP cell and PC3 lines. Results are presented at 48 and 72 hours of incubation, either in untreated or in Permixon treated (44 and 88 μg/ml) conditions. The graphic shows the reduction of cell growth according to the absorbance of treated and untreated cultures (XTT Cell Proliferation kit Sigma-Aldrich).

Article Snippet: LNCaP and PC3 cell lines were obtained from Interlab Cell Line Collection (ICLC) (Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy).

Techniques: Incubation

Journal: Journal of Inflammation (London, England)

Article Title: Effect of Serenoa repens (Permixon®) on the expression of inflammation-related genes: analysis in primary cell cultures of human prostate carcinoma

doi: 10.1186/1476-9255-10-11

Figure Lengend Snippet: Detection of caspase-3 in apoptotic cells. Immunocytochemistry (immunofluorescence) with anti-cleaved caspase3 by rhodamine indirect labelling. The upper panels are PC3 untreated ( a ) and treated ( b ) with LSESr for 16 hours. The lower panels are primary cells line untreated ( c ) and treated ( d ) with Permixon® for 16 hours. Untreated cells show no labelling with the anti-cleaved caspase-3 antibody and TRITC anti IgG. LSERSr treated cells show (arrows) labelling with anti-cleaved caspase-3.

Article Snippet: LNCaP and PC3 cell lines were obtained from Interlab Cell Line Collection (ICLC) (Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy).

Techniques: Immunocytochemistry, Immunofluorescence

Journal: Journal of Inflammation (London, England)

Article Title: Effect of Serenoa repens (Permixon®) on the expression of inflammation-related genes: analysis in primary cell cultures of human prostate carcinoma

doi: 10.1186/1476-9255-10-11

Figure Lengend Snippet: GADPH, IL-6, CCL-5 (RANTES), CCL-2, COX-1, COX-2 and iNOS RT-PCR products separated on a 2% agarose gel followed by ethidium bromide staining in PC3, LNCaP and primary cells lines. In PC3 (panel 1) and LNCaP (panel 2) analysis was performed at 8, 24 and 72 hours of incubation whereas in primary cell lines (panel 3) at 16 hours of interval. Here we report results obtained from one prostatectomy, representative of 40 independent experiments. The experiment was performed in untreated and LSESr treated (44 μg/ml and 88 μg/ml in PC3 and LNCaP and only 44 μg/ml in primary cultures) conditions. UN = untreated; LS = LSESr treated; T = primary cells culture derived from PC nodules; Ct = primary cells culture derived from normal prostate tissue surrounding the tumor.

Article Snippet: LNCaP and PC3 cell lines were obtained from Interlab Cell Line Collection (ICLC) (Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy).

Techniques: Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, Incubation, Derivative Assay

Journal: Journal of Inflammation (London, England)

Article Title: Effect of Serenoa repens (Permixon®) on the expression of inflammation-related genes: analysis in primary cell cultures of human prostate carcinoma

doi: 10.1186/1476-9255-10-11

Figure Lengend Snippet: NF-κB detection. Immunofluorescence of p65 NF-κB in PC3 ( a, b, c ) and LNCaP ( d, e, f ) cell lines. In untreated condition ( a, d ) NF-κB is 100% detected in the cytoplasm of cells (arrows). After LSESr (44 μg/ml) treatment ( b, e ) = 24 hours and ( c, f ) = 48 hours of incubation more than 30% of NF-κB translocated at nuclear level (arrows).

Article Snippet: LNCaP and PC3 cell lines were obtained from Interlab Cell Line Collection (ICLC) (Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy).

Techniques: Immunofluorescence, Incubation