Nuclear condensation of cells incubated for 5 days with tamoxifen (Tx) and R115,777

<p><b>Copyright information:</b></p><p>Taken from "Farnesyl-transferase inhibitor R115,777 enhances tamoxifen inhibition of MCF-7 cell growth through estrogen receptor dependent and independent pathways"</p><p>Breast Cancer Research 2005;7(6):R1159-R1167.</p><p>Published online 21 Nov 2005</p><p>PMCID:PMC1410750.</p><p>Copyright © 2005 Dalenc et al.; licensee BioMed Central Ltd.</p> Cells were fixed and stained with the DNA intercalating agent DAPI (left) and nuclei were examined by fluorescence microscopy. DAPI-stained nuclei were counted as normal or condensed nuclei (right). One randomly selected field is presented for each treatment. For three independent experiments, quantification of at least 200 nuclei was performed for each treatment. Error bars indicate the mean values ± standard error of the mean. Cells were fixed and stained for the detection of the caspase cleavage product of cytokeratin 18 by immuno-histochemistry. Floating and adherent cells are represented in cytospin preparations. These preparations were fixed and stained with monoclonal antibody M30 CytoDeath. The proportion of positive cells (brown) was calculated as a percentage of the total number of cells. All determinations were performed in triplicate with at least 400 cells being counted. Error bars indicate the mean values ± standard error of the mean. Cells were fixed and stained for the detection of the caspase cleavage product of cytokeratin 18 by FACS. Floating and adherent cells were harvested, fixed and stained with the fluoresein-conjugated monoclonal antibody M30 CytoDeath and analysed using flow cytometry. The proportion of positive cells was calculated as a percentage of the total number of cells. Data are representative of one to three independent experiments; intra-assay variations wer

Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection-2

to the human immunoglobulins sequences (imgt). Sequences highlighted in red vary from the amino-acid sequence of scFv C1. Underlined sequences correspond to CDR. Asterisks indicate the missing amino-acid that would generate the usual CDR.<p><b>Copyright information:</b></p><p>Taken from "Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection"</p><p>http://www.biomedcentral.com/1472-6750/8/34</p><p>BMC Biotechnology 2008;8():34-34.</p><p>Published online 31 Mar 2008</p><p>PMCID:PMC2323369.</p><p></p

Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection-7

sequence of bacterial pectate lyase that mediates secretion into the periplasmic space; : variable fragment of the heavy chain; : light chain; : 6 histidine-tag; : myc-tag; amber stop codon; : portions of the N- and C-term of phage capside protein pIII; and : primers used for sequencing the Vand Vdomain. Schematic construction of vectors encoding soluble scFv C1 and scFv C1-N1N2. The amber stop codon between the scFv and gene III in pHEN 2 was removed by mutagenesis (middle construct). The C-terminal portion of pIII was removed in the final pHEN C1-N1N2 vector (bottom construct), first by PCR amplification of pHEN C1-pIII, introducing an EcoRI site after N2, and subsequently by cloning the NcoI and EcoRI digested PCR product into the linearized pHEN C1-pIII plasmid at the NotI and EcoRI sites. : 6 histidine-tag; M: myc-tag.<p><b>Copyright information:</b></p><p>Taken from "Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection"</p><p>http://www.biomedcentral.com/1472-6750/8/34</p><p>BMC Biotechnology 2008;8():34-34.</p><p>Published online 31 Mar 2008</p><p>PMCID:PMC2323369.</p><p></p

Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection-0

sequence of bacterial pectate lyase that mediates secretion into the periplasmic space; : variable fragment of the heavy chain; : light chain; : 6 histidine-tag; : myc-tag; amber stop codon; : portions of the N- and C-term of phage capside protein pIII; and : primers used for sequencing the Vand Vdomain. Schematic construction of vectors encoding soluble scFv C1 and scFv C1-N1N2. The amber stop codon between the scFv and gene III in pHEN 2 was removed by mutagenesis (middle construct). The C-terminal portion of pIII was removed in the final pHEN C1-N1N2 vector (bottom construct), first by PCR amplification of pHEN C1-pIII, introducing an EcoRI site after N2, and subsequently by cloning the NcoI and EcoRI digested PCR product into the linearized pHEN C1-pIII plasmid at the NotI and EcoRI sites. : 6 histidine-tag; M: myc-tag.<p><b>Copyright information:</b></p><p>Taken from "Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection"</p><p>http://www.biomedcentral.com/1472-6750/8/34</p><p>BMC Biotechnology 2008;8():34-34.</p><p>Published online 31 Mar 2008</p><p>PMCID:PMC2323369.</p><p></p

Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection-6

recombinant RhoA and RhoB loaded with either GTP or GDP were incubated with C1-N1N2 fixed on Ni-beads. An irrevelant scFv was used as control. Complexes on beads were resolved by SDS-PAGE and immunobloted with anti-RhoA and anti-RhoB. Western Blot is representative of 2 independent experiments. Immunofluorescence shows that scFv C1-N1N2 specifically binds to activated HeLa cells. Suspension containing scFv C1-N1N2 was incubated with GDP-loaded RhoA(and ) or GTPγS-preloaded RhoA beads (). Twenty-four hours after seeding, HeLa cells were serum-starved for 48 h and activated with 10% SVF and EGF (100 ng/ml) for 1 hour. Cells were fixed, permabilized and incubated with supernatants from scFv C1-N1N2-Rho incubation and anti-c-myc FITC conjugate secondary antibody. ) Non-activated HeLa cells incubated with the antibody scFv C1-N1N2 preincubated with GDP-loaded RhoA beads, ) EGF-activated HeLa cells incubated with the antibody scFv C1-N1N2 preincubated with GDP-loaded RhoA beads ) EGF-activated HeLa cells incubated with the antibody scFv C1-N1N2 preincubated with GTPγS-loaded RhoA beads. () Non-activated HeLa cells incubated with the commercial Rhoa antibody, () EGF-activated HeLa cells incubated with the commercial RhoA antibody, () EGF-activated HeLa cells incubated with irrelevant scFv (anti-tyroglobulin). Pictures are representative of 2 independent experiments.<p><b>Copyright information:</b></p><p>Taken from "Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection"</p><p>http://www.biomedcentral.com/1472-6750/8/34</p><p>BMC Biotechnology 2008;8():34-34.</p><p>Published online 31 Mar 2008</p><p>PMCID:PMC2323369.</p><p></p

Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection-4

ScFvs were resolved on 12,5% SDS-PAGE, immunoblotted with c-myc antibody, and visualized by enhanced chemiluminescence as described in Methods. (2) scFvs from crude extract were analysed for binding to GST-RhoA and GST-RhoAQ63L protein immobilized on an ELISA plate. Bound scFvs were detected with horseradish peroxydase-labeled anti-c-myc using TMB as substrate. Results are expressed as absorbance at 480 nm. Graphs are representative of 6 experiments, each performed in duplicate.<p><b>Copyright information:</b></p><p>Taken from "Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection"</p><p>http://www.biomedcentral.com/1472-6750/8/34</p><p>BMC Biotechnology 2008;8():34-34.</p><p>Published online 31 Mar 2008</p><p>PMCID:PMC2323369.</p><p></p

Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection-1

RhoB (white columns) and GST-RhoBQ63L (black columns) protein immobilized on an ELISA plate. Bound phages were detected with horseradish peroxydase-labeled anti-M13 using TMB as substrate. Helper phage was used as a control. Results are expressed as absorbance at 480 nm. : ratio of absorbance of binding to RhoBQ63L to absorbance of binding to RhoB. : Selectivity of C1 phages on WT and activated Q63L form of Rho. 10clones of C1 (black columns) and control (white columns) phage were analyzed for binding to GST-RhoA, RhoB and RhoC, both wild type (WT) and Q63L forms, immobilized on a glutathione ELISA plate. Bound phages were detected with horseradish peroxydase-labeled anti-M13 using TMB as substrate. Results are expressed as absorbance at 480 nm. Concentrations of GST-Rho proteins in each well were monitored by anti-GST (not shown). The graph is representative for 3 experiments, and each binding assay was performed in duplicate.<p><b>Copyright information:</b></p><p>Taken from "Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection"</p><p>http://www.biomedcentral.com/1472-6750/8/34</p><p>BMC Biotechnology 2008;8():34-34.</p><p>Published online 31 Mar 2008</p><p>PMCID:PMC2323369.</p><p></p

Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection-3

100 μM of GDP and GTPγS at 37°C. 10clones of C1 phages were incubated with loaded (GTP or GDP) GST-RhoA-bound beads. Bound phages were detected with horseradish peroxydase-labeled anti-M13 using TMB as substrate. Results are expressed as absorbance at 480 nm. The graph is representative of 2 independents experiments. GST-RhoA, B and C () and GST-RhoB, Rac1 and Cdc42 () were loaded with 200 μM of GDP or GTPγS for 30 min and purified on glutathione ELISA plates. 10clones of C1 phages were incubated in each well. Bound phages were detected with horseradish peroxydase-labeled anti-M13 using TMB as substrate. Results are expressed as absorbance at 480 nm. Amount of GST protein was quantified with goat anti-GST antibody followed by horseradish peroxydase-labeled anti-goat (not shown). The graph is representative of 3 experiments, each binding assay performed in duplicate. : Specific radioactivity binding of [S] GTPγS on RhoB, Rac1 and Cdc42. GST-RhoB, Rac1 and Cdc42 were loaded with 20 nM [S] GTPγS in the presence (non specific binding) or not (total binding) of 200 μM unlabeled GTP for 30 minutes at 37°C and purified on gluthatione ELISA plates. Radioactivity was measured in each well. The difference between the total binding and the non specific binding represent the specific binding. The graph is representative of 2 experiments, each performed in triplicate.<p><b>Copyright information:</b></p><p>Taken from "Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection"</p><p>http://www.biomedcentral.com/1472-6750/8/34</p><p>BMC Biotechnology 2008;8():34-34.</p><p>Published online 31 Mar 2008</p><p>PMCID:PMC2323369.</p><p></p

Immunocytochemical analysis of the effects of prenyltransferase inhibitors on estrogen receptor (ER) α distribution in MCF-7 cells

<p><b>Copyright information:</b></p><p>Taken from "Prenylation inhibitors stimulate both estrogen receptor α transcriptional activity through AF-1 and AF-2 and estrogen receptor β transcriptional activity"</p><p>Breast Cancer Research 2004;7(1):R60-R70.</p><p>Published online 8 Nov 2004</p><p>PMCID:PMC1064103.</p><p>Copyright © 2004 Cestac et al., licensee BioMed Central Ltd.</p> Cells, deprived of estradiol (E2) for 7 days, were treated or not with FTI-277 (10 μM or dithiothreitol/dimethylsulfoxide vehicle), and 24 hours later they were stimulated with E2 (5 nM) or ethanol and treated or not with FTI-277 or GGTI-298 (10 μM or 5 μM, respectively, or vehicle). After 24 hours, cells were fixed and stained. One randomly selected field is presented for each treatment. Data were quantified by determining the grey value of both the nucleus and the cytoplasm for each cell counted, as described in Materials and methods. For each experimental condition, six randomly selected fields were analyzed. The total number of cells present in the fields ranged from 250 to 400. Error bars indicate the mean values ± standard error of the mean, and the results are representative of two independent experiments

Effects of prenyltransferase inhibitors on estrogen response element-dependent luciferase activity in HELN cells, transfected with estrogen receptor (ER) α or its mutants

<p><b>Copyright information:</b></p><p>Taken from "Prenylation inhibitors stimulate both estrogen receptor α transcriptional activity through AF-1 and AF-2 and estrogen receptor β transcriptional activity"</p><p>Breast Cancer Research 2004;7(1):R60-R70.</p><p>Published online 8 Nov 2004</p><p>PMCID:PMC1064103.</p><p>Copyright © 2004 Cestac et al., licensee BioMed Central Ltd.</p> Cells, deprived of estradiol (E2) for 4 days, were co-transfected with the Renilla luciferase plasmid and either HEG0 (full-length ERα), HEG19 (AF-1-deleted ERα), HEG7 (AF-2-deleted ERα) or pSG5 (empty vector). Five hours after transfection, cells were treated or not with FTI-277 (10 μM or dithiothreitol/dimethylsulfoxide vehicle), and 24 hours later they were stimulated with E2 (5 nM) or ethanol and treated or not with FTI-277 or GGTI-298 (10 μM or 5 μM, respectively, or dithiothreitol/dimethylsulfoxide vehicle). Luciferase activity was quantified 16 hours after E2 addition, as described in Materials and methods. Results are expressed in arbitrary units after normalization. Error bars indicate the mean values ± standard deviation from triplicate experiments, and the results are representative of three independent experiments. Results obtained show that prenylation inhibitors statistically increase or do not statistically increase the luciferase activity on their own compared with control cells (white bars) and statistically increase the luciferase activity induced by E2 compared with the activity induced by E2 alone (grey bars) in HELN cells transfected with ERα or its mutants (* < 0.02)