Fig 2: Schematic model depicting the involvement of phosphorylated eIF4E and eIF2a in stress-responsive, uORF-mediated translation regulation. Upon anisomycin treatment, p38, Mnk1 and mTOR signaling pathways converge at eIF4E for the uORFchop-driven translation, with phospho-eIF2a also playing certain role in this regulation. In contrast, under thapsigargin treatment, uORFchop-driven translation depends exclusively on the induced phosphorylation of eIF2a/S51. See text for detailed discussion on the roles of these signaling pathways and their interplay.

Fig 3: Rapamycin treatment represses anisomycin-, but not thapsigargin-induced uORFchop-driven translation. (A) 293T cells were transfected with dual reporter construct (ulR-uORF-Lu) and treated with anisomycin (An, 0.5 µM), thapsigargin (Tg, 1 µM), rapamycin (Ra, 1 µM) or a combination of An + Ra or Tg + Ra. Cell extracts were harvested for Dual-Luciferase Reporter Assay (upper panel, mean ± SD, P < 0.05) and western blot analysis (lower panel). The blots were probed with the indicated antibodies. Anisomycin treatment induced both the protein level and the activated form of mTOR (i.e. phosphorylated mTOR/S2481) and its targets, such as eIF4G, S6K1, and 4E-BP1. (B) Extracts from cells subjected to drug treatment (1 h) as in (A) were immunoprecipitated with anti-eIF4E antibody followed by western blot analysis. The blots were probed with anti-eIF4E, anti-eIF4E/S209 (eIF4E-P), anti-4E-BP1, anti-eIF4G and anti-Mnk1 antibodies. (C) Cells were treated with anisomycin, or with kinase inhibitor targeting either p38, ERK, Mnk or mTOR, in the presence or absence (−) of anysomycin. Cell extracts were harvested for western blot analysis using antibody against CHOP. The asterisk indicates significant increase of this ratio as compared to the control.

Fig 4: ARID1A down-expression promotes EMT process in endometrial cancer context. (A) Representative images at time 0 and 48 h after scratch, of wound-healing assay performed in IK cells infected with lentiviruses carrying sgRNA against ARID1A (lentiCRISPRv2-ARID1A.2 and lentiCRISPRv2-ARID1A.3) (top panel) and quantification of wound closure area between the indicated time (bottom blot). Statistical analysis was performed using one-way ANOVA analysis followed by the Tukey’s multiple comparison. (B) Representative images of nuclear Hoechst staining of transwell invasion assay after the cotton swab in IK cells infected with lentiviruses carrying sgRNA against ARID1A (lentiCRISPRv2-ARID1A.2 and lentiCRISPRv2-ARID1A.3) (upper panel) and quantification of Matrigel® invasive cells (bottom plot). Scale bars: 50 µm. Statistical analysis was performed using one-way ANOVA analysis followed by the Tukey’s multiple comparison. (C) Gene set enrichment analysis (GSEA) was used to uncover signatures associated to cellular adhesion and EMT (Upper) and enriched gene (bottom) from the molecular signatures database TCGA_UCEC. (D) Representative images of immunofluorescence against E-cadherin, ß-catenin, Cytokeratin, Vimentin and Hoechst in IK cells infected with lentiviruses carrying sgRNA against ARID1A (lentiCRISPRv2-ARID1A.2 and lentiCRISPRv2-ARID1A.3). Magnification images of framed regions of the samples are shown. Scale bars: 25 µm. (E) Representative immunoblots showing E-cadherin, ß-catenin, N-cadherin, Vimentin, MMP2, SNAIL and ZEB protein expression in IK cells infected with lentiviruses carrying sgRNA against ARID1A (lentiCRISPRv2-ARID1A.2 and lentiCRISPRv2-ARID1A.3). GAPDH was used as a loading control. (F) Western blot analysis of phosphorylated and total ERK 1/2, SAPK/JNK and p38 a/ß, c-fos and phospho-MNK1 in IK cells infected with lentiviruses carrying sgRNA against ARID1A (lentiCRISPRv2-ARID1A.2 and lentiCRISPRv2-ARID1A.3). GAPDH was used as a loading control. (G) Metastatic growth of stably infected with EGFP-luciferase MFE-296 cells infected with lentiviruses carrying sgRNA against ARID1A (lentiCRISPRv2- and lentiCRISPRv2-ARID1A.3). After selection, 50 × 104 MFE-296 cells were retro-orbitally injected and let grow for 10 days (see the Materials and methods section for further details). Left-up: representative bioluminescence imaging comparing metastasis in lungs 10 days after injection of MFE-296 ARID1A deficient cells and the parental MFE-296 cell line. Left-down: representative lung and lung morphology analysis by hematoxylin and eosin staining (HE). Right, representative graph evaluating lung histology lesions present in indicated group animals. Scale bars: 100 µm. Statistical analysis was performed using fisher’s exact test. Graph values are the mean and error bars represented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001. Results shown are representative of three independent experiments with three technical replicates per experiment. Graphs showing quantification of immunoblotting plots are shown in Fig. S7; E.V., empty vector.

Fig 5: MNK1 and MNK2 expression in non-malignant (GM02184), malignant (DLBCL) cell lines and primary tissue samples.(a) RT–qPCR analysis of MNK1 (black bar) and MNK2 (grey bar) total mRNA in ABC- and GCB-DLBCL cell lines compared with GAPDH mRNA (mean±s.e.m., n=3, *P-value of Student’s t-test <0.05). (b) Western blot analysis of MNK1 and MNK2 protein expression in ABC- and GCB-DLBCL cell lines with GAPDH loading control. Densitometry analysis is shown in Supplementary Fig. 1a. (c,d) RT–qPCR analysis of (c) MNK1 or (d) MNK2 mRNA levels in total RNA extracted from primary tissue microarray preclassified as normal lymph node (LN, black bar), GCB- (white bar) or ABC- (grey bar) DLBCL, relative to (c) MNK1 or (d) MNK2 levels in normal lymph node (mean±s.e.m., n=3, *P-value of Student’s t-test <0.05). (e) Survival-rescue experiment of HLY-1 DLBCL, 48 h after transduction of non-target (NT, black bar) or MNK1 shRNA (M1KD, white bar) in both empty vector (EV) and MNK2 WT (M2WT) overexpressing cells, measured by trypan blue exclusion assay (mean±s.d., n=3, *P-value of Student’s t-test <0.05). (f) Survival-rescue experiment of HLY-1 DLBCL, 48 h after transduction of non-target (NT, black bar) or MNK2 shRNA (M2KD, white bar), in both empty vector (EV) and MNK1 WT (M1WT) overexpressing cells, measured by trypan blue exclusion assay (mean±s.d., n=3, *P-value of Student’s t-test <0.05). (g) Western blot analysis of total and phospho-eIF4E1(S209) as well as MNK1, MNK2 and GAPDH for MNK1 KD rescue experiment from Fig. 1e in HLY-1 expression mutants. Densitometry analysis is shown in Supplementary Fig. 1b. (h) Western blot analysis of total and phospho-eIF4E1(S209) as well as MNK1, MNK2 and GAPDH for MNK2 KD rescue experiments from Fig. 1f in HLY-1 expression mutants. Densitometry analysis is shown in Supplementary Fig. 1c (i) Representative cell cycle analysis from three independent experiments of stable cell lines expressing empty vector, wild-type MNK1 (MNK1) or MNK1 phosphonull mutant (MNK1-AA) in non-malignant B-cell line GM02184 and DLBCL cell lines HLY-1 and Pfeiffer. Full immunoblots are shown in Supplementary Fig. 10.