Journal: BMC Musculoskeletal Disorders

Article Title: The influence of calcitonin gene-related peptide on markers of bone metabolism in MG-63 osteoblast-like cells co-cultured with THP-1 macrophage-like cells under virtually osteolytic conditions

doi: 10.1186/s12891-016-1044-5

Figure Lengend Snippet: RANKL protein expression in particle- and LPS-stimulated MG-63 osteoblasts remains unchanged even upon CGRP treatment. MG-63 osteoblasts were stimulated with LPS (100 ng/ml) or UHMWPE particles (cell-to-particle ratio of 1:500) and treated with CGRP (10 −8 M). Changes in the expression of both full-length (fl) and soluble (s)RANKL protein were assessed by SDS-PAGE and Western Blot. RANKL protein expression as compared to commercially available LNCaP lysate and recombinant sRANKL as external controls and GAPDH as an internal control is exemplarily shown for cells at 6 and 24 h of incubation. The sRANKL band provided a very strong signal even upon low exposition times so that artifacts can be seen in the neighboring lanes

Article Snippet: Equal amounts of protein (20 μg) along with recombinant human soluble (s)RANKL (100 ng; PeproTech, Hamburg, Germany) and LNCaP whole cell lysate (25 μg, sc-2231; Santa Cruz Biotechnology, Dallas, Texas, USA) were separated by 8–16 % tris-glycine SDS-PAGE (Thermo Fisher Scientific, Waltham, Massachusetts, USA) and then electro-blotted to 0.45 μm nitrocellulose membranes (Bio-Rad, Hercules, California, USA).

Techniques: Expressing, SDS Page, Western Blot, Recombinant, Control, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Buxus natalensis (Oliv.) Hutch (Buxaceae) Exhibits Its Anticancer Potential by Stimulating ROS Production and Caspase-p53-BCL-2-Dependent Apoptosis in Hepatocellular Carcinoma and Prostate Cancer Cell Lines

doi: 10.3390/ijms26094173

Figure Lengend Snippet: Cytotoxic effect of Buxus natalensis leaf extracts on HepG2 ( A ), LNCaP ( B ), and Chang liver ( C ). Ten thousand cells were seeded per well for each cell line on 96-well microtiter plates, and the cells were treated for 48 h with different leaf extracts (15.625–500 µg/mL) under standard cell-culture conditions. The cell viability was estimated as a percentage of cells treated with DMSO (0.5%) considered as 100%. B. natalensis hydroethanolic leaf extract (BNHLE); B. natalensis methanolic extract (BNMLE); B. natalensis aqueous leaf extract (BNALE).

Article Snippet: Eight cancer cell lines, namely, human breast carcinoma cells (MCF-7), 4T1 mammary carcinoma cells, human colorectal carcinoma cells (Caco-2), human cervical carcinoma cells (HeLa), human lung carcinoma cells (A549), human hepatocellular carcinoma (HepG2), DU145 (androgen-insensitive), and LNCaP (androgen-sensitive) human prostate cancer cell lines, were obtained from the American Type Culture Collection (ATCC) (Rockville, MD, USA) or the European Collection of Authenticated Cell Cultures (ECACC) (Porton Down, UK).

Techniques: Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: Buxus natalensis (Oliv.) Hutch (Buxaceae) Exhibits Its Anticancer Potential by Stimulating ROS Production and Caspase-p53-BCL-2-Dependent Apoptosis in Hepatocellular Carcinoma and Prostate Cancer Cell Lines

doi: 10.3390/ijms26094173

Figure Lengend Snippet: Colony formation after treatment with B. natalensis hydroethanolic leaf extract (BNHLE). One thousand cells from each cell line HepG2 ( A ), LNCaP ( B ), and Chang liver ( C ) were seeded in 6-well microtiter plates, and exposed to BNHLE for 24 h, followed by the replacement of the culture medium every 3 days for 15 days. The colonies were stained with crystal violet (0.5%) and allowed to dry overnight. Then, 2 mL of acetic acid (33%) was added per well, and the absorbance was read at 550 nm using a SpectraMax iD3 multi-mode microplate reader (Molecular Devices, San Jose, CA, USA). One-way ANOVA combined with Dunnett or Student–Newman–Keuls’s tests were used for data analysis. * means statistically different ( p < 0.05) vs. Ctrl.

Article Snippet: Eight cancer cell lines, namely, human breast carcinoma cells (MCF-7), 4T1 mammary carcinoma cells, human colorectal carcinoma cells (Caco-2), human cervical carcinoma cells (HeLa), human lung carcinoma cells (A549), human hepatocellular carcinoma (HepG2), DU145 (androgen-insensitive), and LNCaP (androgen-sensitive) human prostate cancer cell lines, were obtained from the American Type Culture Collection (ATCC) (Rockville, MD, USA) or the European Collection of Authenticated Cell Cultures (ECACC) (Porton Down, UK).

Techniques: Staining

Journal: International Journal of Molecular Sciences

Article Title: Buxus natalensis (Oliv.) Hutch (Buxaceae) Exhibits Its Anticancer Potential by Stimulating ROS Production and Caspase-p53-BCL-2-Dependent Apoptosis in Hepatocellular Carcinoma and Prostate Cancer Cell Lines

doi: 10.3390/ijms26094173

Figure Lengend Snippet: Intracellular ROS production in LNCaP cells after treatment with B. natalensis hydroethanolic leaf extract (BNHLE). LNCaP cells (10,000 cells/well) were treated for 45 min with 100 µL of 20 µM of DCFH-DA diluted in culture medium containing 2% FBS. Thereafter, these cells were exposed for 24 h to BNHLE (100, and 200 µg/mL), and hydrogen peroxide (H 2 O 2 , 50 µM) or DMSO (0.5%) used as negative control (Ctrl). Images were captured using a Flexacam C1 camera (Leica Microsystems GmbH, Wetzlar, Germany) connected to a fluorescence microscope (Leica Microsystems GmbH, Wetzlar, Germany) using an excitation/emission filter (480/535 nm) on 20× objective ( A ). The cell fluorescence was measured at the wavelengths of 485 nm (excitation) and 535 nm (emission) on a SpectraMax iD3 multi-mode microplate reader (Molecular Devices, San Jose, CA, USA), and intracellular ROS levels were expressed as percentage of negative control cells ( B ). One-way ANOVA combined with Dunnett or Student–Newman–Keuls’s tests were used for data analysis. * means statistically different ( p < 0.05) vs. Ctrl.

Article Snippet: Eight cancer cell lines, namely, human breast carcinoma cells (MCF-7), 4T1 mammary carcinoma cells, human colorectal carcinoma cells (Caco-2), human cervical carcinoma cells (HeLa), human lung carcinoma cells (A549), human hepatocellular carcinoma (HepG2), DU145 (androgen-insensitive), and LNCaP (androgen-sensitive) human prostate cancer cell lines, were obtained from the American Type Culture Collection (ATCC) (Rockville, MD, USA) or the European Collection of Authenticated Cell Cultures (ECACC) (Porton Down, UK).

Techniques: Negative Control, Fluorescence, Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Buxus natalensis (Oliv.) Hutch (Buxaceae) Exhibits Its Anticancer Potential by Stimulating ROS Production and Caspase-p53-BCL-2-Dependent Apoptosis in Hepatocellular Carcinoma and Prostate Cancer Cell Lines

doi: 10.3390/ijms26094173

Figure Lengend Snippet: Caspase luminescence activity in HepG2 ( A , B ) and LNCaP cells ( C , D ) after exposure to B. natalensis hydroethanolic leaf extract (BNHLE). HepG2 and LNCaP cells (10,000 cells/well) were exposed for 18 h to BNHLE and doxorubicin hydrochloride (Doxo, 2 µM) or DMSO (0.5%) used as negative control (Ctrl). Caspase reagents were added in equal volume as the culture medium, and luminescence was read using SpectraMax iD3 multi-mode microplate reader (Molecular Devices, San Jose, CA, USA). Caspase activities were expressed as a percentage (fold change) of cells treated with DMSO (0.5%) used as a negative control. One-way ANOVA combined with Dunnett or Student–Newman–Keuls’s tests were used for data analysis. * means statistically significantly different ( p < 0.05) vs. Ctrl.

Article Snippet: Eight cancer cell lines, namely, human breast carcinoma cells (MCF-7), 4T1 mammary carcinoma cells, human colorectal carcinoma cells (Caco-2), human cervical carcinoma cells (HeLa), human lung carcinoma cells (A549), human hepatocellular carcinoma (HepG2), DU145 (androgen-insensitive), and LNCaP (androgen-sensitive) human prostate cancer cell lines, were obtained from the American Type Culture Collection (ATCC) (Rockville, MD, USA) or the European Collection of Authenticated Cell Cultures (ECACC) (Porton Down, UK).

Techniques: Activity Assay, Negative Control

Journal: International Journal of Molecular Sciences

Article Title: Buxus natalensis (Oliv.) Hutch (Buxaceae) Exhibits Its Anticancer Potential by Stimulating ROS Production and Caspase-p53-BCL-2-Dependent Apoptosis in Hepatocellular Carcinoma and Prostate Cancer Cell Lines

doi: 10.3390/ijms26094173

Figure Lengend Snippet: Expression levels of human p53 and NF-κB-p65 in HepG2 ( A , B ) and LNCaP ( C , D ) after 24 h of treatment with B. natalensis hydroethanolic leaf extract (BNHLE). Cells were treated for 24 h with BNHLE, or DMSO (0.5%) used as negative control (Ctrl). Equal amounts of proteins in cell lysates were used for the quantification of the expression levels of NF-κB-p65 and p53 using commercially available ELISA kits (Cat# E-EL-H1388 and Cat# E-EL-H0910, Elabscience Biotechnology Inc., Houston, TX, USA). One-way ANOVA combined with Dunnett or Student–Newman–Keuls’s tests were used for data analysis. # means statistically significantly different ( p < 0.05) vs. Ctrl.

Article Snippet: Eight cancer cell lines, namely, human breast carcinoma cells (MCF-7), 4T1 mammary carcinoma cells, human colorectal carcinoma cells (Caco-2), human cervical carcinoma cells (HeLa), human lung carcinoma cells (A549), human hepatocellular carcinoma (HepG2), DU145 (androgen-insensitive), and LNCaP (androgen-sensitive) human prostate cancer cell lines, were obtained from the American Type Culture Collection (ATCC) (Rockville, MD, USA) or the European Collection of Authenticated Cell Cultures (ECACC) (Porton Down, UK).

Techniques: Expressing, Negative Control, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Buxus natalensis (Oliv.) Hutch (Buxaceae) Exhibits Its Anticancer Potential by Stimulating ROS Production and Caspase-p53-BCL-2-Dependent Apoptosis in Hepatocellular Carcinoma and Prostate Cancer Cell Lines

doi: 10.3390/ijms26094173

Figure Lengend Snippet: Expression levels of human BAX and BCL-2 in LNCaP ( A , B ) and HepG2 ( C , D ) as well as their respective BAX/BCL-2 ratio ( E , F ) after 24 h of treatment with B. natalensis hydroethanolic leaf extract (BNHLE). Cells were treated for 24 h with BNHLE, or doxorubicin hydrochloride (Doxo, 2 µM) or DMSO (0.5%) used as negative control (Ctrl). Equal amounts of proteins in cell lysates were used for the quantification of the expression levels of BAX and BCL-2 using commercially available BCL-2 Associated X protein and BCL-2 ELISA kits (Cat# EEL030 and Cat# BMS244-3, Thermo Fisher Scientific, Massachusetts, MA, USA), respectively. The BAX/BCL-2 ratio was calculated by dividing the BAX expression level against the BCL-2 level at each tested concentration. One-way ANOVA combined with Dunnett or Student–Newman–Keuls’s tests were used for data analysis. * means statistically significantly different ( p < 0.05) vs. Ctrl.

Article Snippet: Eight cancer cell lines, namely, human breast carcinoma cells (MCF-7), 4T1 mammary carcinoma cells, human colorectal carcinoma cells (Caco-2), human cervical carcinoma cells (HeLa), human lung carcinoma cells (A549), human hepatocellular carcinoma (HepG2), DU145 (androgen-insensitive), and LNCaP (androgen-sensitive) human prostate cancer cell lines, were obtained from the American Type Culture Collection (ATCC) (Rockville, MD, USA) or the European Collection of Authenticated Cell Cultures (ECACC) (Porton Down, UK).

Techniques: Expressing, Negative Control, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Asian Journal of Andrology

Article Title: Growth inhibition mediated by PSP94 or CRISP-3 is prostate cancer cell line specific

doi: 10.1038/aja.2010.56

Figure Lengend Snippet: Pattern of endogenous PSP94 and CRISP-3 expression in different prostate cell lines. (A): Reverse transcription-polymerase chain reaction (RT-PCR) results of the total RNA content that was extracted from the five tested cell lines. The panels from the top to the bottom show amplification of PSP94, CRISP-3 and GAPDH, respectively. Lane 1 is a 100-bp ladder. Lanes 2, 4, 6, 8 and 10 are RT-PCR products for PC3, DU145, LNCaP, RWPE-1 and WPE1-NB26, respectively. Lanes 3, 5, 7, 9 and 11 are PCR reactions for no RT enzyme controls for the same samples. Lane 12 is a water control. (B): Semi-quantitative Western blot analysis for detection of CRISP-3 protein. TCA-precipitated proteins from conditioned medium that was harvested at 48 h (left side) and 72 h (right side) from 5 × 105 PC3 cells, WPE1-NB26 cells and LNCaP cells were subjected to Western blot analysis using an anti-CRISP-3 polyclonal antibody. The presence of two bands at around 30 and 28 kDa in the WPE1-NB26 and LNCaP cells represent glycosylated and unglycosylated CRISP-3 protein, respectively.

Article Snippet: Human PC3, DU145 and LNCaP were obtained from the National Centre for Cell Sciences, Pune, India.

Techniques: Expressing, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Amplification, Control, Western Blot

Journal: Asian Journal of Andrology

Article Title: Growth inhibition mediated by PSP94 or CRISP-3 is prostate cancer cell line specific

doi: 10.1038/aja.2010.56

Figure Lengend Snippet: Secreted recombinant PSP94 interacts with endogenous CRISP-3. (A): Western blot analysis for detection of PSP94HA. Cell lysate and TCA-precipitated proteins derived from the conditioned medium from PC3 cells that were transiently transfected with either empty vector (V) or a PSP94 expression construct (P) were probed with anti-HA antibody. (B): Localization of PSP94HA by immunofluorescence. PC3 cells that were transiently transfected with empty vector (i) and a PSP94 expression construct (ii) as well as LNCaP cells transiently transfected with empty vector (iii) and a PSP94 expression construct (iv) were first probed with polyclonal anti-HA antibody and then with FITC-labelled secondary antibody. Bars = 20 μm. (C): Effect of ectopically expressed PSP94 on endogenous CRISP-3. TCA-precipitated proteins from vector-transfected and PSP94 expression construct-transfected WPE1-NB26 and LNCaP cells were subjected to Western blot analysis 48 h after transfection. The upper panel was probed with anti-CRISP-3 antibody and the lower panel was probed with anti-HA antibody. (D): Co-immunoprecipitation of PSP94 and CRISP-3 from the conditioned medium of vector-transfected and PSP94 expression construct-transfected WPE1-NB26 cells. PSP94HA was immunoprecipitated using anti-HA agarose-conjugated beads. The upper portion of the Western blot was probed with anti-CRISP-3 antibody and the lower portion was probed with anti-HA antibody. The lanes marked with input show the total amount of CRISP-3 and PSP94HA that was immunoprecipitated from the conditioned medium.

Article Snippet: Human PC3, DU145 and LNCaP were obtained from the National Centre for Cell Sciences, Pune, India.

Techniques: Recombinant, Western Blot, Derivative Assay, Transfection, Plasmid Preparation, Expressing, Construct, Immunofluorescence, Immunoprecipitation

Journal: Asian Journal of Andrology

Article Title: Growth inhibition mediated by PSP94 or CRISP-3 is prostate cancer cell line specific

doi: 10.1038/aja.2010.56

Figure Lengend Snippet: Ectopic expression of PSP94 has an inhibitory effect on cell growth in a cell line specific manner. (A): A representative composite picture of the crystal violet staining from the clonogenic survival assay. A single well (of the six-well plate) is shown for each cell line (PC3, WPE1-NB26 and LNCaP) following transfection with empty vector, pcDNA3.1+ (left wells) or PSP94 expression construct (right wells). At 24 h after transfection, the cells were split and stable clones were selected on G418. Clones obtained after two weeks of selection were stained and counted. (B): Effect of the over-expression of PSP94 on the clonogenic survival of PC3, WPE1-NB26 and LNCaP cells. For each cell line, the number of clones obtained in the vector-transfected wells was considered to represent 100% survival. The number of clones obtained in the PSP94 expression construct-transfected wells was used to calculate the percentage of surviving cells with respect to the empty vector-transfected cells. The assay was repeated at least three times for each cell line. The graph is the average of three independent observations and is plotted as the mean percentage survival ± SE. The observed reduction in the percentage survival of WPE1-NB26 and LNCaP cells was statistically significant when compared with PC3 cells (*P < 0.05, compared with PC3 cells; one-way ANOVA).

Article Snippet: Human PC3, DU145 and LNCaP were obtained from the National Centre for Cell Sciences, Pune, India.

Techniques: Expressing, Staining, Clonogenic Cell Survival Assay, Transfection, Plasmid Preparation, Construct, Clone Assay, Selection, Over Expression

Journal: Asian Journal of Andrology

Article Title: Growth inhibition mediated by PSP94 or CRISP-3 is prostate cancer cell line specific

doi: 10.1038/aja.2010.56

Figure Lengend Snippet: Cell line specific growth-inhibitory effect caused by CRISP-3 over-expression. (A): Expression of recombinant CRISP-3 was detected by Western blot analysis using anti-CRISP-3 antibody. TCA-precipitated proteins from the conditioned medium of PC3 cells that were transiently transfected with CRISP-3 expression construct (Lane 1) showed the presence of glycosylated and unglycosylated CRISP-3 similar to the positive control (Lane 3) that contained TCA-precipitated proteins from the conditioned medium of WPE1-NB26 cells. TCA-precipitated proteins from the conditioned medium of PC3 cells transfected with empty vector (Lane 2) as well as untransfected RWPE-1 (Lane 4) served as a negative control. (B): A representative composite picture of the crystal violet staining from the clonogenic survival assay. A single well (of the six-well plate) is shown for each cell line (PC3, WPE1-NB26 and LNCaP) that was transfected with either the empty vector, pEFIRES-P (left wells), or the CRISP-3 expression construct (right wells). At 24 h after transfection, the cells were split and stable clones were selected on puromycin. Clones obtained after 2 weeks of selection were stained and counted. (C): Effect of over-expression of CRISP-3 on the clonogenic survival of PC3, WPE1-NB26 and LNCaP cells. The percentage survival with respect to empty vector was calculated as described above. The assay was repeated at least three times for each cell line. The graph is the average of three independent observations and is plotted as mean percentage survival ± SE. The reduction in percentage survival of PC3 and WPE1-NB26 cells was statistically significant when compared with LNCaP cells (*P < 0.05, compared with LNCaP; one-way ANOVA).

Article Snippet: Human PC3, DU145 and LNCaP were obtained from the National Centre for Cell Sciences, Pune, India.

Techniques: Over Expression, Expressing, Recombinant, Western Blot, Transfection, Construct, Positive Control, Plasmid Preparation, Negative Control, Staining, Clonogenic Cell Survival Assay, Clone Assay, Selection

Journal: Asian Journal of Andrology

Article Title: Growth inhibition mediated by PSP94 or CRISP-3 is prostate cancer cell line specific

doi: 10.1038/aja.2010.56

Figure Lengend Snippet: The growth-inhibitory effect of CRISP-3 is not modulated by PSP94. (A): A representative composite picture of the crystal violet staining from the clonogenic survival assay for the stable clones PC3-D4, PC3-A5 (PSP94-expressing clones) and PC3-A1v (empty vector-transfected clone). A single well (of the six-well plate) is shown for each clone that was transfected with either empty vector, pEFIRES-P (left wells), or the CRISP-3 expression construct (right wells). At 24 h after transfection, cells were split and selected on puromycin. Clones obtained after 2 weeks of selection were stained and counted. (B): The effect of over-expression of CRISP-3 on the clonogenic survival of PC3-D4, PC3-A5 and PC3-A1v. The percentage survival with respect to empty vector was calculated as described above. The assay was repeated at least three times for each cell line. The graph is the average of three independent observations and is plotted as the mean percentage survival ± SE. The reduction in percentage survival of all three clones was statistically significant when compared with LNCaP (*P < 0.05, compared with LNCaP shown in Figure 5B; one-way ANOVA).

Article Snippet: Human PC3, DU145 and LNCaP were obtained from the National Centre for Cell Sciences, Pune, India.

Techniques: Staining, Clonogenic Cell Survival Assay, Clone Assay, Expressing, Plasmid Preparation, Transfection, Construct, Selection, Over Expression

Journal: Asian Journal of Andrology

Article Title: Growth inhibition mediated by PSP94 or CRISP-3 is prostate cancer cell line specific

doi: 10.1038/aja.2010.56

Figure Lengend Snippet: Cell line specific effect of the ectopic expression of PSP94 or CRISP-3.

Article Snippet: Human PC3, DU145 and LNCaP were obtained from the National Centre for Cell Sciences, Pune, India.

Techniques: Expressing, Clone Assay

Journal: Oncotarget

Article Title: Establishment of prostate cancer spheres from a prostate cancer cell line after phenethyl isothiocyanate treatment and discovery of androgen-dependent reversible differentiation between sphere and neuroendocrine cells

doi: 10.18632/oncotarget.8440

Figure Lengend Snippet: A. Representative bright-field images of LNCaP cell spheres cultured in RPMI-1640 medium with 10% FBS. From left to right are floating 3D-sphere, 400x magnifications; fluorescent image of a GFP-labeled LNCaP cell sphere, 400x magnifications; and an adherent sphere, 200x magnifications. B. The left is an image of H & E stained paraffin embedded LNCaP cells; the center is a cross section of a sphere, paraffin embedded and H& E stained; and the right is cross section of a sphere, paraffin embedded and immuno-stained for Ki67 expression. The dark spots are the Ki67 positive cells. Images are 400x magnifications.

Article Snippet: An LNCaP cell line labeled with green fluorescent protein (GFP-LNCaP) was purchased from AntiCancer, Inc. (San Diego, California).

Techniques: Cell Culture, Labeling, Staining, Expressing