Fig 2: Eif2ak4 depletion results in increased AIEC intracellular replication and enhanced AIEC-induced production of cytokines and chemokines. Wild-type (WT) MEFs and MEFs depleted for Eif2ak4 (eif2ak4-/-) were infected with AIEC LF82 for 6 h (B), 12 h (D), 24 h (E) or the indicated time (A, C). In parallel, MEFs were treated with KRB for 4 h. (A) Western blot analysis for the activation of the EIF2AK4-EIF2A-ATF4 pathway. (B) qRT-PCR analysis for mRNA expression levels of ATF4 target genes, including Trib3, Atf3, Ddit3 and Asns. (C) Intracellular LF82 number counted on LB agar plates. (D) Representative confocal micrographs of cells infected with LF82-GFP. Actin cytoskeleton was stained with Rhodamine-phalloidin (red). Nuclei were stained with Hoechst (blue). Bars: 5 µm. (E) Secreted IL6 and CXCL1 amounts in cell culture supernatant quantified by ELISA. Data are means ± SEM of 6 replicates and are representative of 3 independent experiments. *, P < 0.05; **, P = 0.005; ***, P = 0.001.

Fig 3: EIF2A promotes cell survival during paclitaxel treatment in vitro. A, Indicated cells were transfected with EIF2A siRNA, and followed by paclitaxel treatment. Cells were lysed at indicated time points for Western blot assays of EIF2A, HSPA5, p-EIF2S1, EIF2S1 and cleaved caspase 3. B and E, Indicated cells transfected with indicated siRNA were incubated with 100 nmol/L paclitaxel for 48 h. Cell viability was measured by CCK-8 assay. Percentage of cell survival is represented as mean ± SD from 3 independent experiments (n = 3, mean ± SD). **P < 0.01, Student's t test. C, indicated cells stained with Annexin-V and PI, and analysed by FACS. Bars indicate mean values ± SD of three experiments. **P < 0.01. D, MDA-MB-231 and BT-549 transfected with indicated siRNA. Western blots were performed with indicated antibodies

Fig 4: The EIF2AK4-EIF2A-ATF4 pathway is activated in noninflamed CD and deactivated in inflamed CD. Total proteins and RNA were extracted from the terminal ileal biopsy specimens from normal controls or patients with CD (with normal appearance [noninflamed CD] or with macroscopic inflammation [inflamed CD]). (A) Western blot analysis for the activation of the EIF2AK4-EIF2A-ATF4 pathway is shown in the left panel. Quantification of band intensity is shown in the right graphs. (B) qRT-PCR analysis for mRNA expression levels of the known ATF4 target genes (TRIB3, ATF3, DDIT3 and ASNS) and the autophagy genes shown to be regulated by ATF4 in this study (SQSTM1, MAP1LC3B, BECN1, ATG3 and ATG7). Data are represented as scatter dot plots; each dot shows one case number and the bars show the median. Data were analyzed by the nonparametric Mann-Whitney test. *, P < 0.05; **, P = 0.005; ***, P = 0.001.

Fig 5: Cysteine-mediated redox regulation is involved in various biological processes and pathways.a A pie chart showing the distribution of the 1537 proteins that exhibit at least one redox-sensitive cysteine with respect to their functions in different organelles. Subcellular localizations for C. elegans proteins are retrieved from the UniProt knowledge base. b, c Biological processes and pathways enriched in the C. elegans redoxome, indicated by GO (b, blue) and KEGG (c, red) analysis. d, e Representative XICs showing changes in IPM-labeled peptides from LET-363/mTOR (d) and DAF-16/FOXO (e). The profiles for light- and heavy-labeled peptides are shown in red and blue, respectively. The average RT/C values calculated from two biological replicates are displayed below individual XICs. f Fluorescent images and quantification g showing nuclear accumulation of DAF-16::GFP after 5 h of t-BOOH treatment (mean ± SEM, n = 3 experiments, at least 100 worms per condition). **P = 0.0100; Two-tailed Student’s t-test. Scale bar = 100 µm. h A schematic diagram showing that various stress stimuli promote eIF2a phosphorylation by GCN-2. i Representative XICs showing changes in IPM-labeled peptides from GCN-1. j Representative western blots and quantification k showing that eIF2a phosphorylation in response to 30 min t-BOOH exposure is impaired in gcn-1(-) animals (mean, n = 2 experiments). Lysates were loaded onto two different gels for detection with different antibodies, and blots were processed in parallel.