Fig 2: EIF2A promotes cell survival during paclitaxel treatment in vivo. A, B and C, Size and weight of xenograft tumours formed by MDA-MB-231 derivatives including a doxycycline-inducible shRNA targeting EIF2A and scramble shRNA. Each group was treated as indicated. Data are mean ± SEM **P < 0.01. D, Immunohistochemical analyses of the expression of EIF2A and p-EIF2S1 in xenograft tumours as described above. E, Immunohistochemical analyses of patient samples for p-EIF2S1 levels in patients. Top: paracentesis specimens before treatment. Bottom: surgical specimens after paclitaxel-based neoadjuvant chemotherapy. F, Immunohistochemical analysis was graded on a scale of 1-3 according to staining intensity. Specimens form 30 patients participated in the statistic. Paired t test was used for calculating statistical significance. G, Probability of relapse-free survival in 3955 breast cancer patients stratified on low (black) versus high (red) expression levels of indicated genes was obtained from Kaplan-Meier Plotter/breast cancer (http://www.kmplot.com)

Fig 3: Early transcriptional regulation of inflammatory response precedes a translational repression of the translational equipment. (A) Over-representation analysis (ORA) of GO Biological Process terms for up- or down-regulated genes in RNA-seq, Ribo-seq or TE over all time-points. For each time-point, DRGs were selected by comparison to the non-infected condition. (B-F) Induction of inflammation and of the integrated stress response by Lm infection of LoVo cells. The variation of RNA (B, D) and RFP (C, E) levels was quantified for selected NF-?B transcriptional target genes related to inflammation (B, C) or genes involved in the integrated stress response (D, E) during infection. Data represent DESeq normalized read counts from three independent experiments and error bars indicate standard deviation. (F) The phosphorylation status of the mTOR substrate S6K1, and of the target of ISR eIF2 kinases eiF2a were assessed by immunoblotting in cells infected for 2, 5 or 10 h by wild-type Lm or by an Lm strain where the hlyA gene encoding LLO had been deleted (?hlyA). Treatments by tunicamycin and rapamycin were used, respectively, as an inducer of the ISR through endoplasmic reticulum stress, and as a repressor of mTOR activity. (G) Transcripts sharing similar TE profiles over time were clustered by fuzzy clustering. For each cluster, only genes having more than 70% membership are displayed. (H) Functional categories were assigned to each cluster by ORA of GO Biological Process terms.

Fig 4: Related to Figure 3A: Phase separated fraction of G3BP1(WT) in the presence of 5-Loop and polyadenylated 5-Loop RNAs (n = 8 FOV). B: Fluorescence images of GFP-G3BP1(WT)-RNA condensates formed with polyadenylated and Cy5-labeled 5-Loop RNA. Scale bar, 5 µm. Condensates in A-B were formed in the presence of 1% PEG-20K and 75 ng/µl RNA. C: Quantification of the soluble fraction of RNA remaining after tangle formation (mean and SD from three independent measurements). D: Fluorescence immunostaining of oxidatively stressed U2OS wild-type cells and U2OS G3BP1/2 KO cells. G3BP1 (green) and eIF3? (magenta) are shown. Scale bars, 10 µm. E: Quantification of the number of eIF3?-positive SGs per cell in U2OS wild-type and in U2OS G3BP1/2 KO cells (mean and SD, n = 184 - 259 cells). F: Translation levels in U2OS cells (WT or G3BP1/2 KO) upon oxidative stress and recovery from stress. Translation levels were assessed by puromycin incorporation. Immunoblots against puromycin, p-eIF2a (to follow stress kinetics) and eIF2a (loading control) are shown. Harringtonine (Harr.) was used to prove specific incorporation of puromycin into nascent polypeptide chains. On the right, the normalized translation levels upon stress and recovery are shown (mean and SD from three independent experiments). G: EMSA to determine the apparent binding affinity of G3BP1(WT), G3BP1(?RG) and G3BP1(?NTF2) to A(60)-Cy5 RNA. Black arrow points toward free A(60)-Cy5 RNA. * indicates shifted RNA species due to G3BP1 binding. H: EMSA testing for the competition of unlabeled A(60) and poly(A) (500-4000 nt) RNAs for G3BP1(WT) binding to the A(60)-Cy5 probe. From left to right: Lane1: without G3BP1, Lane 2: without competitor, Lane 3: with unlabeled A(60) RNA as competitor, Lane 4: with unlabeled poly(A) as competitor. Black arrow points toward free A(60)-Cy5 RNA. * indicates shifted RNA species due to G3BP1 binding.

Fig 5: Induction of the ISR is critical for resistance to paclitaxel. A, Immunoblots of lysates from blank (without transfected) and EIF2S1 WT (shEIF2S1 + widetype EIF2S1) and EIF2S1 S51A (shEIF2S1 + S51A mutated EIF2S1). B, Indicated cells were incubated with 100 nmol/L paclitaxel for 4 hours and Western blots were performed. C, Indicated cells were incubated with 100 nmol/L paclitaxel and 50 µmol/L Trolox for 48 h. Cell viability was performed by CCK-8 assay. Percentage of cell survival is represented as mean ± SD from three independent experiments (n = 3, mean ± SD). **P < 0.01, Student's t test. D, Indicated cells stained with Annexin-V and PI, and analysed by FACS. Bars indicate mean values ± SD of three experiments. **P < 0.01