Fig 2: RITA’s effects on clonogenicity and p53 activation depend on phosphorylation of eIF2alpha.a Western blot analysis of indicated proteins using extracts from HT1080 WT and HT1080 KI (eIF2aS51A) cells treated with vehicle (DMSO) or 1 µM RITA for 8 h. b Crystal violet staining of HT1080 WT and HT1080 KI cells treated with increasing concentrations of RITA. c Western blot analysis of indicated proteins using extracts from HT1080 WT and KI cells treated with vehicle (DMSO) or increasing concentrations of RITA for 8 h. d Schematic of RITA’s p-eIF2a dependent effects on p53 reactivation, protein synthesis and anti-cancer activity

Fig 3: The maternal LP diet activates the protein levels of p-eIF2a (a) and ATF4 (b) in the female offspring. Total eIF2a was used to normalise the phosphorylation of eIF2a, and actin was used to normalise the protein levels of ATF4. Values are reported as the mean values with their standard error of mean, n 8. *P < 0·05 by post hoc Bonferroni test. p-eIF2a, phosphorylated eukaryotic translation initiation factor 2a; ATF4, activating transcription factor 4. F-C, female offspring born to the dams of control diet; F-LP, female offspring born to the dams of low-protein diet; M-C, male offspring born to the dams of control diet; M-LP, male offspring born to the dams of low-protein diet. (a) , C; , LP. (b) , C; , LP.

Fig 4: Loss of function of JNK3 promotes dysregulation of normal metabolic function. Protein levels were detected. A Endoplasmic reticulum-related [PERK, P-PERK (Thr980), EIF2, P-EIF2 (Ser51), ATF4, IRE1a and P-IRE1a (Ser724)] and B PTP1B and P-PTP1B (Ser50). Results were represented as bar graphs and differences between groups was analyzed using ANOVA and Tukey’s (n = 4 per experimental group). Significance was represented as follows: *p < 0.05, **p < 0.01 and ***p < 0.001

Fig 5: Gemcitabine induces the integrated stress pathway in pancreatic cancer cells. (a) AsPC-1 and PANC-1 cells were incubated with 0.5 µM ISRIB or 10 µM Gem for indicated times and protein lysates were analyzed by immunoblotting for phospho-eIF2, total eIF2, and ATF4 using specific antibodies. ERK2 served as loading control. (b) Empty vector or GADD34 expression plasmid DNA was transfected into PANC-1 cells, which were incubated 24 h later with 10 µM Gem. Protein lysates were analyzed by immunoblotting. (c) PANC-1 cells were incubated for 36 h with 0.5 µM ISRIB or 10 µM Gem and protein lysates were analyzed by immunoblotting for ATF4, ATF3, GADD34, and CHOP using specific antibodies. ERK2 served as loading control. (d) PANC-1 cells were incubated with 0.5 µM ISRIB or 10 µM Gem for 24 h. ATF4, CHOP, ATF3, and GADD34 mRNA levels were measured using qRT-PCR. Relative actin mRNA levels were used for normalization. Data are the means±S.D. from three experiments. *P<0.05, **P<0.001 compared with control, ##P<0.01 compared with ISRIB, and $P<0.05 compared with Gem+ISRIB. (e) MEFs were incubated for 6 h with 10 µM Gem in the absence and presence of ISRIB. As a positive control, MEFs were also incubated for 6 h with thapsigargin (0.1 µM). (f) Wild-type MEFs or MEFs mutated for eIF2a at serine 51 (to alanine) were incubated with 10 µM Gem for indicated time point and analyzed by immnuoblotting. Panels (a, b, d and e) show representative data from three independent experiments