siRNA mediated knockdown of eIF4E in NIH 3T3 cells.

<p>NIH 3T3 cells were transiently transfected with an siRNA against murine eIF4E or with a control siRNA, 4E-T-inv (scrambled sequence of human 4E-T), for 48 hr. Cells were lysed, and protein extracts were subjected to SDS-PAGE, followed by western blot analysis. The RNAi-mediated knockdown was repeated three times.</p

eIF4E induction causes an increase in the recruitment of a subset of mRNAs to polysomes.

<p>A) Total and polysomal (24 fractions) RNA from induced (−tet for 5 hr) and uninduced (+tet for 5 hr) 3T3-tTA-eIF4E cells was reverse transcribed into cDNA. Primers for BI-1, survivin, MIF, L23, L34, L9, S17 and actin were used to assess mRNA levels. Amplified PCR bands from the polysomal fractions were quantified using NIH Image, and absolute values were plotted. B) The effect of eIF4E induction on L34 mRNA distribution was assessed by northern blotting. Polysomal RNA was isolated from induced (−tet for 5 hr) and uninduced (+tet for 5 hr) 3T3-tTA-eIF4E cells and fractionated into 12 fractions (for purpose of detection). The RNA was loaded on an agarose denaturing gel and transferred to a nitrocellulose membrane. Membranes were probed with radiolabeled murine L34 and actin probes. Bands were quantified using NIH Image, and absolute values were plotted.</p

eIF4E induction stimulates the translation of a subset of mRNAs.

<p>A) Western blotting of extracts from 3T3-tTA versus 3T3-tTA-eIF4E cells after eIF4E induction (0 to 24 hr) and from NIH 3T3 cells and MEFs that constitutively express HA-eIF4E or vector alone. eIF4E induction was determined by using anti-HA and anti-eIF4E antibodies. Fold increase at the 24 hr time point was determined using NIH Image. B) Western blotting experiments were performed as described in (A). These experiments were repeated three times using three different sets of whole-cell extracts.</p

Overexpression of phosphomimetic eIF4E-S209D does not affect proliferation but increases clonogenic cell survival.

<p>A, Western blot analysis showed similar, moderate levels of Myc-tagged exogenous eIF4E-S209D or-S209A and endogenous eIF4E expression in MDA-MB-231 and HaCaT cell lines. B, MTT cell proliferation assays revealed no significant difference in cell growth between GFP—or eIF4E-mutant–expressing cells under normal conditions. C, Crystal violet staining of clonogenic colony formation assays clearly indicate an increase in both colony number and size upon eIF4E-S209D expression compared with GFP control cells, which is reflected by a statistically significant increase in the total number of stained cells as measured by overall crystal violet absorbance. A moderate but not statistically significant increase in eIF4E-S209A–expressing cells was noted. Graphs depict the overall cell growth as measured in a crystal violet absorption assay. The survival and growth advantage after expression of eIF4E-S209D is highly significant in both HaCaT and MDA-MB-231 cells treated with arsenite for 90 minutes to induce oxidative stress before being plated. D, Graphs representing the percentage of the number of colonies after arsenite treatment compared to normal conditions in each case. Arsenite treatment clearly decreases the number of colonies in both cell lines. * = P<0.05, ** = P<0.01 and *** = P<0.001, compared to control, n = 3. (AU: Absorbance Units)</p

eIF4E-S209D rapidly modulates protein translation after arsenite treatment.

<p>Polysome analysis of MDA-MB-231 cells showed that cells expressing eIF4E-S209D, but not-S209A or GFP, induced a peak in the 80S fraction 2 hours after arsenite treatment, indicating translational stalling. Twelve hours after recovery from arsenite treatment, the 80S peak in S209D-expressing cells was more moderate compared with that of S209A- or GFP-expressing cells, indicating a slightly higher level of protein synthesis. B, m7-GTP pull-down assays in normal conditions and 2 hours after arsenite treatment indicate that eIF4E-S209D strongly associates with the mRNA cap in a complex with eIF4G in the recovery period after arsenite treatment, which may allow re-initiation of protein synthesis.</p

eIF4E induction protects cells against ER-mediated apoptosis.

<p>A) 3T3 cells that stably express HA-eIF4E were treated with 5 µM ionomycin for different periods, fixed and stained with propidium iodide. The percentage of apoptosis was quantified by flow cytometry (triplicates were pooled to generate the s.d.) B) 3T3-tTA and 3T3-tTA-eIF4E cells were seeded at 75% confluency and cultured for 8 hr. Tetracycline containing medium was then replaced by a tetracycline free medium for 16 hr to induce eIF4E expression. Uninduced cells were cultured for the same period without removal of tetracycline. Cells were treated with ionomycin and processed as described in (A). C) 3T3-tTA and 3T3-tTA-eIF4E cells were seeded and cultured with or without tetracycline as described in (B). Cells were then cultured for 24 hr in complete medium±5 µM ionomycin. Protein extracts were resolved by SDS-PAGE and transferred to membranes, which were immunoblotted with anti–caspase 3 and anti–caspase 12. eIF4E expression was also examined by immunoblot; elevated eIF4E levels were only detected in 3T3-tTA-eIF4E induced cells (−tet).</p

Induction of eIF4E in NIH 3T3 fibroblast cells and microarray analysis.

<p>A) eIF4E overexpression was induced by culturing 3T3-tTA-eIF4E cells in a tetracycline free medium. Immunoblots for eIF4E and β-actin were performed. B) A characteristic fractionation profile of 3T3-tTA and 3T3-tTA-eIF4E cells is depicted. Absorbance at 254 nm was monitored. C) Fractions from 3T3-tTA and 3T3-tTA-eIF4E (induced) cells were analyzed on a denaturing agarose gel to visualize the 18S and 28S rRNAs. D) The experimental design used for microarray analysis is shown.</p

MDA-MB-231 and HaCaT cells show increased resistance to stress after overexpression of phosphomimetic eIF4E.

<p>A, MDA-MB-231 and HaCaT cells were subjected to either arsenite (NaAsO₂), nutrient starvation, or cisplatin (CDDP) treatment, and cell viability was measured by an MTT assay after 24, 48, and 72 hours. In all cases, eIF4E-S209D significantly increased cell viability. B, Apoptotic activity measured by a caspase-3/-7 luminescence assay. Significant activation of caspase-3/-7 activity was observed following arsenite treatment in eIF4E-S209A– and GFP–expressing cells, which was completely prevented by eIF4E-S209D. * = P<0.05, ** P<0.01 and *** = P<0.001 compared to control, n = 3.</p

Selective increase in protein synthesis in cells expressing eIF4E-S209D.

<p>A, Western blotting analysis showed that eIF4E-S209D was able to maintain the expression levels of some proteins, such as cyclin D, after stress caused by arsenite treatment (compared to S209A- or GFP-expressing cells). Other proteins showed reduced levels or no changes. B, Treatment with actinomycin D for 6 hours in normal conditions and 2 hours after stress indicated that the selective advantage of eIF4E-S209D in maintaining protein expression was due to post-transcriptional events. C, Treatment with cycloheximide after arsenite treatment showed that cyclin D and Mcl-1 are subjected to translational regulation by eIF4E-S209D, rather than modulation of protein stability. D, Quantitative PCR analysis of polysomal RNA indicated that eIF4E-S209D after stress permits a dramatic increase in actively translated cyclin D1 (expressed as a fraction of total mRNA).</p

Sequence composition influences eIF4E responsiveness.

<p>Top row: median fold change of four groups of sequences corresponding to the four possible nucleotides at each position in the alignment around a) cap region (nt 1–20), b) start region (positions −19…20 with position 1 being the first nt of the coding region), c) stop region (positions −19…20 with position 1 being the first nt of the 3′UTR). Blue: A, red: C, green: G, black: U. Bottom row: Negative decadic logarithm of the Kruskal-Wallis test p-value as a function of the sequence position. The statistical test is applied at each alignment column to the fold change values of the four groups mention above. Note that because the Kruskal-Wallis test is not defined for completely conserved alignment columns, the start and stop codon regions are skipped (Figures a)–c)). The eIF4E overexpression data set consisting of 11387 mRNAs was used to generate the plots (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004868#s2" target="_blank">Materials and Methods</a>).</p