Figure 1.
Effect of GSI treatment on the expression of eIF6 and eIF4E.
The different T-cell leukemia derived cell lines were treated with 5 µM DAPT or control DMSO for 16h. Total RNA was isolated and expression levels of eIF6 (A) or eIF4E mRNA (B) were estimated by RT-PCR. The intensity of the relevant bands was normalized with respect to the control GAPDH. The histograms represent the average of three independent experiments.
Figure 2.
Analysis of RBP-Jk-binding sites in the human eIF6 promoter.
(A) Shown is a diagram of the human eIF6 gene 5′-flanking region upstream from the first exon. The positions of putative RBP-Jk-binding sites (sites A–B) and of the PCR primers used in ChIP experiments (FR1 and FR2) are indicated (black arrows). (B) EMSA were performed using nuclear extracts of the indicated cells and the labeled promoter fragment probes. The arrow in all three panels indicates the position of the shifted site A fragment; the asterisk in the third panel indicates the position of the supershift resulting from the addition of anti-RBP-jk antibodies. (C) ChIP assays. Chromatin from Jurkat cell extracts was immunoprecipitated with anti-Notch1 antibodies (N1 Ab) or with control rabbit IgG. The IPs were analyzed by PCR using primers specific for the indicated promoter regions of eIF6 or for the Notch-binding region of the Hes1 promoter as the positive control.
Figure 3.
(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 Renilla 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).
Figure 4.
eIF6 expression in stably-transfected A2780 ovarian cancer cells.
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.
Figure 5.
eIF6 over-expression does not significantly affect cell cycle.
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.
Figure 6.
Over-expression of eIF6 enhances migration and invasivity of ovarian cancer cells.
(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.
Table 1.
List of the primers used for the cloning of different constructs.