eIF6 over-expression does not significantly affect cell cycle.

<p>FACS analysis of cell-cycle distribution of control (A) and eIF6-over-expressing (B) A2780 cells grown for 72 h in the absence (top) or in the presence (bottom) of 75 µM DAPT.</p

eIF6 expression in stably-transfected A2780 ovarian cancer cells.

<p>eIF6 expression in a pool of A2780 cells stably transfected with the pcDNA3-eIF6 plasmid was analyzed by RT-PCR (A) and western blotting (B) as indicated. The intensity of the eIF6 RNA and protein bands was quantified relative to β-actin and β-tubulin, respectively, using the ImageJ software. The results represent the average of three independent experiments. (C) Analysis of the polysomal profiles of A2780/eIF6 and control cells by density gradient centrifugation. The areas under the polysomal peaks were quantified using the ImageJ software.</p

Over-expression of eIF6 enhances migration and invasivity of ovarian cancer cells.

<p>(A, B) Migration assay: A2780/eIF6 and control cells were treated with DAPT 75 µM or DMSO for 36 h then seeded in the upper side of migration chambers. The cells migrated to the lower chambers after 36 h of incubation were stained with crystal violet dye. (C, D) Invasivity assay: cells were treated with DMSO or DAPT 75 µM for 36 hours and seeded in the upper side of invasion chambers. After 36 h, cells migrated in the lower chamber were stained. The total stained area in the lower chambers was estimated using the Image-J software. The histograms in (B) and (D) represent the average of three independent experiments. P values estimating the statistical significance of the observed experimental variations between different data sets (control cells with and without GSI; eIF6 cells with and without GSI; control and eIF6 cells without GSI; control and eIF6 cells with GSI) are shown for both cell migration and invasion experiments.</p

List of the primers used for the cloning of different constructs.

<p>The reaction sites used for the cloning are underlined.</p

Luciferase reporter assay.

<p>(A) Schematic representation of the region of the human eIF6 promoter containing two putative RBP-jk-binding sites (sites A-B). Different tracts of the promoter were cloned upstream of the luciferase gene in the pGL3 Basic luciferase plasmid obtaining the constructs indicated as FL, FR1, FR2 and FR3. (B) Luciferase assay. NIH-3T3 cells were cotransfected with <i>Renilla</i> luciferase plasmid and one of the reporter plasmids shown in A in the presence (black columns) or the absence (grey columns) of a plasmid expressing human Notch-1 (N1). (C) NIH-3T3 cells co-transfected with the FL plasmid and the N1 plasmid were further transfected with increasing amounts (0,25-0,5-1 µg) of a plasmid expressing a dominant-negative form of RBP-jk (RBP-jk DN).</p

Functional analysis of the translation factor aIF2/5B in the thermophilic archaeon -6

<p><b>Copyright information:</b></p><p>Taken from "Functional analysis of the translation factor aIF2/5B in the thermophilic archaeon "</p><p></p><p>Molecular Microbiology 2007;65(3):700-713.</p><p>Published online 01 Aug 2007</p><p>PMCID:PMC1976387.</p><p>© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd</p>synthesized in a translation systems programmed with increasing amounts of aIF2/5B mRNA and fixed amounts of a leaderless (α-fucosidase) or a leadered (104) mRNA. Lane 1: 10 pmol of α-fuc or 10 pmol of 104 mRNA in the absence of aIF2/5B mRNA; Lanes 2–4: same with 10, 20 and 40 pmol, respectively, of aIF2/5B mRNA. Bottom. Curve obtained by quantifying with a Molecular Dynamics Phosphoimager the radioactivity retained in the bands as shown in the top panel. The relative increment on the -axis is the ratio between radioactivity values in the absence of added aIF2/5B mRNA and values measured in the presence of the indicated amounts of aIF2/5B mRNA

Functional analysis of the translation factor aIF2/5B in the thermophilic archaeon -8

<p><b>Copyright information:</b></p><p>Taken from "Functional analysis of the translation factor aIF2/5B in the thermophilic archaeon "</p><p></p><p>Molecular Microbiology 2007;65(3):700-713.</p><p>Published online 01 Aug 2007</p><p>PMCID:PMC1976387.</p><p>© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd</p

Functional analysis of the translation factor aIF2/5B in the thermophilic archaeon -2

<p><b>Copyright information:</b></p><p>Taken from "Functional analysis of the translation factor aIF2/5B in the thermophilic archaeon "</p><p></p><p>Molecular Microbiology 2007;65(3):700-713.</p><p>Published online 01 Aug 2007</p><p>PMCID:PMC1976387.</p><p>© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd</p

Interaction of native and chimeric IF2 factors with archaeal and bacterial ribosomal subunits

<p><b>Copyright information:</b></p><p>Taken from "Functional analysis of the translation factor aIF2/5B in the thermophilic archaeon "</p><p></p><p>Molecular Microbiology 2007;65(3):700-713.</p><p>Published online 01 Aug 2007</p><p>PMCID:PMC1976387.</p><p>© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd</p> The ribosome-bound fraction of the various proteins was determined as described in . Circle: recombinant aIF2/5B; diamond: recombinant IF2; triangle: BaSu1 chimera; square: BaSu2 chimera

Functional analysis of the translation factor aIF2/5B in the thermophilic archaeon -4

<p><b>Copyright information:</b></p><p>Taken from "Functional analysis of the translation factor aIF2/5B in the thermophilic archaeon "</p><p></p><p>Molecular Microbiology 2007;65(3):700-713.</p><p>Published online 01 Aug 2007</p><p>PMCID:PMC1976387.</p><p>© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd</p