Influence of eIF4GII on gene translation, cell proliferation and the senescent phenotype.

<p>(A) 72 h after transfecting HeLa cells with either siRNA Ctrl or siRNA targeting eIF4GII, cell lysates were fractionated through 10–50% linear sucrose gradients, and the polysome distribution profiles were analyzed. (B) HeLa cells were transfected as described in (A) and nascent protein synthesis was assessed by incorporation of ³⁵S-labeled amino acids. The ³⁵S-amino acid incorporation was quantified and presented as percentage of signal intensity relative to control transfection (graph.). (C) Protein levels of miR-520c-3p target genes were measured by Western blotting 72 h after transfection of HeLa cells with either control siRNA or eIF4GII siRNA. GAPDH was used as a loading control. (D) Cells were transfected as described in (A) and the mRNA levels of selected miR-520c-3p target mRNAs and housekeeping GAPDH mRNA in each gradient fraction were measured by RT-qPCR and plotted as a percentage of the total mRNA levels in each sample. Data represent the average of three independent experiments showing similar results. (E) 72 h after transfection HeLa and Farage cells with either siRNA Ctrl or targeting eIF4GII siRNA, cell numbers were counted with a hemocytometer. (F) Six days after transfection as described in (E) cells were tested for apoptosis. (G) 72 h after transfection as described in (E) HeLa cells were stained with PI and subjected to cell cycle analysis. (H) β-galactosidase activity was measured in HeLa cells six days after transfection as described in (F) (I) Six days after transfections as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004105#s4" target="_blank">Materials and Methods</a>, the levels of p16, p53, and HuR in HeLa and Farage cells were assessed by Western blot analysis. GAPDH served as a loading control. Data are representative of at least three independent experiments. In the graphs (B), (E), (F) and (G) the means and SEM are shown. * p<0.05.