Fig 2: eIF3e silencing increases GBM cell radiosensitization. (A) GO enrichment analysis of the mRNAs that are upregulated after eIF3e silencing and plotted with REVIGO. Size of the rectangles reflects the P-value (log10). (B, C) Plating efficiency assays measuring the surviving fraction in LN18 (B) or U251 (C) cells treated with control (siScr) or eIF3e (si3e) siRNAs and submitted to a radiation dose scale. Data are presented as mean values ± SEM of three independent experiments; **P < 0.005, ***P < 0.0005 (paired t-test). DEF for U251 and LN18 cells is measured as follows: for the same biological effect (here surviving fraction of log 0.1), the dose on the curve radiation alone (here siScr) is divided by the dose on the curve radiation + treatment (here si3e)). A DEF >1 means that the treatment is functioning as a radiosensitizer.

Fig 3: eIF3e expression in different GBM histological regions and in GSCs. (A) Relative mRNA expression for eIF3e in different tumor regions from the Ivy GBM database. Data are presented as mean values ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001, NS: non-significant (Benjamini–Hochberg test) (i). Representative IHC images for eIF3e expression in microvascular proliferating (ii) and pseudopalisading regions (iii). (B) Genome-wide transcriptome microarray analysis (GSE67089) showing that eIF3e was among the most upregulated translation factors in glioma spheres (i). mRNA expression analysis (GSE67089) of eIF3e in the mesenchymal (Mes) and the proneural (PN) subtypes of GSCs compared with normal neurosphere (ii). *P < 0.05 and ***P < 0.001, by one-way ANOVA followed by Dunnett’s post-hoc test. n = 6 for PN, n = 3 for Mes and n = 1 for normal neurosphere.

Fig 4: eIF3e drives selective mRNA translation of mRNAs involved in GBM tumor progression and response to treatments. (A) Polysome profile of U251 cells treated with control (siScr) or eIF3e (si3e) siRNAs. mRNA was isolated from fractions containing three ribosomes (box) and analyzed in parallel with cytoplasmic mRNA using microarrays (see the ‘Materials and Methods’ section). The peaks corresponding to the 40S, 60S and 80S ribosomal subunits and polysomes are indicated, as well as NP, LP and HP fractions. (B) Microarray data analysis using a Venn diagram showing the overlap between cytosolic mRNAs and translationally regulated mRNAs. The number of eIF3e activated or repressed mRNAs in each group is indicated. (C) Validation of the genome-wide polysome profiling in (A) by RT-qPCR analysis from pooled NP and HP fractions using specific primers for the indicated mRNAs and quantification by analyzing the ratio HP/NP [error bars ± SEM of three experiments; *P < 0.05, ***P < 0.0005 (paired t-test)]. (D) Western blot analysis of the indicated proteins in U251 and LN18 cells treated with control (siScr) or eIF3e (si3e) siRNAs. (E) Ratio of Renilla/Firefly luciferase activities (Rluc/Fluc) (ii) determined using U251 cells treated with control (siScr) and eIF3e (si3e) siRNAs, followed by co-transfection with Rluc and Fluc mRNA reporters depicted in (i) [error bars ± SEM of three independent experiments; *P < 0.05 (paired t-test)].

Fig 5: eIF3e regulates HIF translation in glioblastoma cells. (A) Western blot analysis of HIF-1α and HIF-2α in GBM cell lines treated with control (siScr) and eIF3e (si3e) siRNAs, with or without a proteasome inhibitor treatment (MG132 at 5 μM for 2 h) (i) and with or without a hypoxia mimetic treatment (CoCl2 at 150 μM for 12 h) (ii). Shown is a representative result from two to four experiments. (B) Annexin V/PI staining and flow cytometry analysis for GBM cells treated as in (A). Data are presented as mean values ± SEM of four independent experiments; *P < 0.05, **P < 0.01, ***P < 0.001 (paired t-test). (C) Quantitative RT-qPCR using specific primers for HIF-1αand HIF-2α mRNAs extracted from U251 cells treated with control (siScr) or eIF3e (si3e) siRNAs. Data are plotted relatively to B2M mRNA amounts and are presented as mean values ± SEM of four to six independent experiments. NS: non-significant (Wilcoxon test). (D) RT-qPCR analysis using specific primers for HIF-1α and B2M mRNAs extracted from translationally inactive (NP) or active (including LP and HP) fractions obtained after fractionation on a sucrose gradient of cytoplasmic extracts of U251 cells treated with control (siScr) or eIF3e (si3e) siRNAs. Shown is a representative distribution of two independent experiments.