Fig 2: Correlation between GCN2, PERK, JNK and FOXO3 expression and activity in response to epirubicin and palclitaxel treatment. The epirubicin sensitive MCF-7 and resistant MCF-7EpiR cells were either left untreated or treated with 1 µM epirubicin for the times shown. B. The paclitaxel sensitive MCF-7 and resistant MCF-7TaxR were either left untreated or treated with 10 nM paclitaxel for the times indicated. Whole-cell protein lysates were analysed by western blotting using the antibodies recognising the proteins indicated. Molecular weight markers are shown. The protein expression levels of P-FOXO3 (T32) (95 kDa), FOXO3 (95 kDa), FOXM1 (110 kDa) and ER stress molecules including P-GCN2 (160 kDa), GCN2 (220 kDa), P-PERK (140 kDa), PERK (140 kDa), P-eIF2a (38 kDa), eIF2a (38 kDa), p27Kip1 (27 kDa) and ß-Tubulin (55 kDa) were investigated. C. Parallel RT-qPCRs were performed for studying GCN2, FOXO3, PERK, FOXM1, p27Kip1 mRNA expression following treatment with epirubicin. D. GCN2, FOXO3, PERK, FOXM1, p27Kip1 mRNA expression after treatment with paclitaxel was determined by RT-qPCR. Three technical repeats were performed in one experiment, and the data were normalised to L19 and showed as means ± S.E.M. The expression trends of mRNA species between MCF-7 and MCF-7EpiR or c MCF-7TaxR cells are compared using 2-way ANOVA (Significant ***p < 0.001, for all mRNA species, respectively). One of the three independent repeats is shown.

Fig 3: Quantitative analysis of protein synthesis in primary neuron culture. (A) Normalized ratios of mCherry/GFP fluorescence levels in cell cultures transfected by plasmids with wt or ?1–7 5'UTR of PKM? in the presence/absence of picrotoxin (PTX). (B) Examples of dendrites used for counting. (C) Normalized ratios of mCherry/GFP fluorescence levels in cell cultures transfected by plasmids with wt or ?1–7 5'UTR of PKM? in the presence/absence of Na2AsO3. (D) Western blot analyzes of the primary culture of rat neurons treated/untreated by PTX. Antibodies raised against eIF2a and eIF2aP were used for detection of non-phosphorylated and phosphorylated forms of eIF2a, respectively. (E) Quantitative analysis of the western blots. eIF2a/eIF2aP ratios normalized to total protein (per line). The error bars represent the standard error of mean, (n = 3).

Fig 4: Graphical representation of findings from HT-1080 cells. A, In basal conditions, N92I-RAC1 promotes increased p-MEK1/2 and p-ERK1/2 and p-EIF2S1. Overexpression of ATF4 contributes to p-EIF2S1 homeostasis. B, Upon RAC1 knockdown, reduced p-MEK1/2 and p-ERK1/2 leads to reduced cell proliferation. There is a loss of basal p-EIF2S1 which is not rescued by the concomitant loss of ATF4 expression. C, ER-stress in untransfected cells leads to phosphorylation of EIF2S1 which is quickly attenuated by the N92I-RAC1-driven overexpression of ATF4. N92I-RAC1-dependent p-MEK1/2 and p-ERK1/2 promotes cell survival. D, Loss of constitutive ATF4 expression in RAC1-depleted cells leads to hyper-phosphorylation of EIF2S1 by PERK in ER-stressed cells because negative feedback is attenuated. Reduced pMEK1/2 and p-ERK1/2 causes increased sensitivity to stress. Bold arrows represent comparison with corresponding untransfected cells. Red arrows represent phosphorylation, blue arrows represent expression. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig 5: Effect of hepatic autophagy inactivation on markers of the hepatic UPR in critically ill mice. The ratio of hepatic p-eIF2alpha over eIF2alpha protein expression (a), hepatic mRNA expression of atf4 (b), xbp1s (c), hspa5 (d), dnajb9 (e), creb3l3 (f), calr (g) and pdia4 (h) are shown for healthy pair-fed WT mice (white box plots), healthy pair-fed KO mice (dotted box plots), critically ill WT mice (gray box plots) and critically ill KO mice (hatched box plots) at day 1 and day 3. Box plots depict medians with interquartile ranges (IQR) and whiskers are drawn to the furthest point within 1.5 × IQR from the box. n = 10–16 per group. p-eIF2alpha: phosphorylated eukaryotic initiation factor 2 alpha; eIF2alpha: eukaryotic initiation factor 2 alpha; atf4: activating transcription factor 4; xbp1s: spliced form of x-box binding protein 1; hspa5: heat shock protein family A member 5; dnajb9: dnaJ heat shock protein family member B9; creb3l3: cAMP responsive element binding protein 3-like 3; calr: calreticulin; pdia4: protein disulfide isomerase family A member 4. KO: Mx1-Cre + Atg7 F/F mouse; WT: Mx1-Cre - Atg7 F/F mouse.