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: Nucleic Acids Research

Article Title: REST mediates androgen receptor actions on gene repression and predicts early recurrence of prostate cancer

doi: 10.1093/nar/gkt921

Figure Lengend Snippet: Analysis of ARORs by ChIP. ( A ) Quantitative PCR analysis of AR binding to promoter region of ARE– and RE-1–containing genes in LNCaP cells treated or not with DHT for 2 h. All gene names are provided by genenames.org. Students t -test was used to analyze the statistical significance of the differences observed on DHT treated versus control samples (* P < 0.05 and ** P < 0.01). ( B ) Heat map illustrating changes in expression on androgen or bicalutamide treatment for genes whose promoters bind AR in ChIP analysis. Data were obtained from GEO database ( http://www.ncbi.nlm.nih.gov/geo/ ). The experiments accession numbers are indicated ( C ) ChIP microarray analysis. The number of regions containing binding sites for the indicated transcription factors within AROR (dark gray) or a set of control regions that did not bind the AR in the ChIP microarray analysis, denoted as controls (light gray). Only the TFBS, with a statistically significant ( P < 0.05, χ2 analysis) overrepresentation within AROR, are shown. The transcription factor binding sites are named using JASPAR similarity matrix names as follows: REST, repressor element-1 silencing transcription factor; PPARG, peroxisome proliferation activated receptor gamma; AR, androgen receptor; NFκB, nuclear factor of kappa light polypeptide gene enhancer in B cells; D-Vit, vitamin D receptor; ESR1, estrogen receptor 1; MIZF, histone H4 transcription factor; bZIP911, ELK4, ETS domain proteins; E2F1, E2F transcription factor; Su(H), suppressor of hairless; NR2F1, nuclear receptor subfamily 2, group F, member 1; MAX MYC, Myc associated factor X; RREB1, Ras-responsive element binding protein 1; T, T box protein; TEAD1, transcriptional enhancer factor TEF1; and NR2F1, nuclear receptor subfamily 2, group F, family 1. ( D ) ChIP analysis of REST binding to promoter region of ARE and RE-1 containing genes in LNCaP cells treated or not with DHT for 2 h. The statistical significance was analyzed using Students t -test (* P < 0.01).

Article Snippet: Eight male athymic Balb/c nude mice (Beijing HFK Bio-Technology Co. Ltd.) were injected subcutaneouslly with 2 × 10 6 LNCaP cells suspended in 0.1 ml of Matrigel (BD Biosciences), half of the mice were castrated when tumors reached 10 mm and the tumors were allowed to grow back and later transplanted orthotopically into the prostate ventral lobe of intact or castrated Balb/c mice.

Techniques: Real-time Polymerase Chain Reaction, Binding Assay, Expressing, Microarray

Journal: Nucleic Acids Research

Article Title: REST mediates androgen receptor actions on gene repression and predicts early recurrence of prostate cancer

doi: 10.1093/nar/gkt921

Figure Lengend Snippet: In situ proximity ligation assays. ( A ) Representative images of REST/AR co-localization in situ visualized by the proximity ligation assays. LNCaP cells were stained with REST and/or AR antibodies as indicated and subjected to proximity ligation assays to measure their co-localization in situ , visualized by the appearance of high-intensity fluorescent spots at the 634 nm wavelength. Two different antibodies recognizing the AR and raised from different species were used as positive controls. Analyses performed in the absence of one of the primary antibodies were used as negative controls. ( B ) The co-localization of AR and REST was analyzed by the quantification of the number of fluorescent spots localized in the nucleus of individual cells (α, P < 0.0001).

Article Snippet: Eight male athymic Balb/c nude mice (Beijing HFK Bio-Technology Co. Ltd.) were injected subcutaneouslly with 2 × 10 6 LNCaP cells suspended in 0.1 ml of Matrigel (BD Biosciences), half of the mice were castrated when tumors reached 10 mm and the tumors were allowed to grow back and later transplanted orthotopically into the prostate ventral lobe of intact or castrated Balb/c mice.

Techniques: In Situ, Ligation, Staining

Journal: Nucleic Acids Research

Article Title: REST mediates androgen receptor actions on gene repression and predicts early recurrence of prostate cancer

doi: 10.1093/nar/gkt921

Figure Lengend Snippet: Androgens regulate REST function. ( A ) LNCaP cells were starved from steroids and then treated with DHT for 24 h. RT-PCR was used to measure expression levels of four known REST target genes: BDNF, Grin2a, NTRK3 and Synapsin1. All differences were statistically significant (* P < 0.01). ( B ) In a pulse chase experiment, LNCaP cells were labeled with 35 S-methionine and treated for 2 and 4 h with androgen when REST was immunoprecipitated and analyzed by autoradiography. ( C ) LNCaP cells were transfected with two siRNA targeting either REST or the AR, followed by measurements of the mRNA levels of the indicated genes. Asterisks indicate statistically significant differences between the cells transfected with control siRNA and those targeting AR or REST (* P < 0.05, ** P < 0.01). Western blot shows protein levels of REST and AR after REST and AR knockdown. ( D ) A luciferase reporter construct driven by the synapsin 1 gene promoter region containing the REST response element (RE-1) and a variant in which the N-terminal half region of the RE element was deleted was used to assess REST-mediated repression in the cells transfected with siRNAs targeting the REST and AR in the presence and absence of androgens. Significant differences in the comparison between luciferase activity in the cells transfected with siControl and Syn-Luc and the other experimental groups are denoted by * P < 0.05 or ** P < 0.01. Significant differences between the control group–transfected Syn-luciferase mutant and the rest of the groups are marked with α, P < 0.01. ( E ) Measurement of REST and AR binding to chromatin regions within the promoters of BDNF, Syn1 and Grin2a genes containing an RE-1 or to the ARE in a PSA gene enhancer. The influence of DHT treatment on REST and AR chromatin binding was also measured. DNA quantization was performed by q-PCR. Means differences between IgG control ChIP and those performed with the indicated antibodies (AR or REST) were compared by Student’s t -test (* P < 0.01 and ** P < 0.05). The statistical differences between DHT treated and untreated samples was also analyzed (α denotes a P < 0.05) ( F ) Measurement of REST, AR and β-TRCP levels in LNCaP cells with the indicated treatments.

Article Snippet: Eight male athymic Balb/c nude mice (Beijing HFK Bio-Technology Co. Ltd.) were injected subcutaneouslly with 2 × 10 6 LNCaP cells suspended in 0.1 ml of Matrigel (BD Biosciences), half of the mice were castrated when tumors reached 10 mm and the tumors were allowed to grow back and later transplanted orthotopically into the prostate ventral lobe of intact or castrated Balb/c mice.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Pulse Chase, Labeling, Immunoprecipitation, Autoradiography, Transfection, Western Blot, Luciferase, Construct, Variant Assay, Activity Assay, Mutagenesis, Binding Assay

Journal: Nucleic Acids Research

Article Title: REST mediates androgen receptor actions on gene repression and predicts early recurrence of prostate cancer

doi: 10.1093/nar/gkt921

Figure Lengend Snippet: REST regulates NE differentiation in LNCaP cells. ( A ) Protein levels of NE marker chromogranin A and REST in cells depleted of androgens for the indicated times in the presence and absence of DHT. ( B ) mRNA levels of the REST target genes: Grin2a and NTRK3 and NE marker chromogranin A in same experiment as above. ( C ) NE differentiation was quantified by analyzing the length of cytoplasmic extensions processes during androgen deprivation for 7 days or in cells grown in the presence of androgen (+DHT) but transfected with siRNAs targeting REST or the AR. Statistical analysis was performed using the Wilcox ranked test; asterisk denotes statistically significant differences ( P < 0.001) with the siControl +DHT group, while α denotes significant differences ( P < 0.001) in comparison with control nontransfected cells −DHT. ( D ) mRNA levels of CgA in REST and AR knockdowns (* P < 0.05, Student’s t -test). ( E ) Protein and mRNA levels of chromogranin A after 72- and 96-h treatment with AR antagonist MDV3100. ( F ) Chromogranin A and REST levels in LNCaP orhotopic xenograft grown in castrated or intact mice.

Article Snippet: Eight male athymic Balb/c nude mice (Beijing HFK Bio-Technology Co. Ltd.) were injected subcutaneouslly with 2 × 10 6 LNCaP cells suspended in 0.1 ml of Matrigel (BD Biosciences), half of the mice were castrated when tumors reached 10 mm and the tumors were allowed to grow back and later transplanted orthotopically into the prostate ventral lobe of intact or castrated Balb/c mice.

Techniques: Marker, Transfection