Fig 2: Analysis of ER stress in response to erastin treatment.(A) Western blot analysis of erastin (5 µM, 7 hr)—sorafenib (10 µM, 12 hr)—and thapsigargin (10 ng/ml, 6 hr)-treated cells reveals changes associated with ER stress including phosphorylation of eIF2alpha and up-regulation of ATF4 protein levels relative to unphosphorylated forms and total protein respectively. *: non-specific band in the ATF4 blot. (B) Splicing of XBP-1 mRNA analyzed by PCR and agarose gel electrophoresis. Treatment conditions are as in (A). (C) Viability of HT-1080 cells over time treated with DMSO or erastin ± actinomycin D or cycloheximide (CHX), as indicated. Cell counts were measured using a Vi-Cell analyzer. (E and F) CHAC1 mRNA levels measured in HT-1080 cells in response to the indicated erastin analogs. (E) AE, PE and MEII were reported previously in Yang et al. (2014). Results presented in A and B are representative of three independent experiments. Data shown in (D and E) is the average of three independent biological replicates, and in (C and F) the average of two replicates.DOI: http://dx.doi.org/10.7554/eLife.02523.007

Fig 3: NNT (upper row), ITAC (lower row). Stained tissue sections using antibodies against EIF2S1, EIF5A and EIF6 in tissue micro arrays (TMAs) at different resolutions.

Fig 4: Correlations between PERK and FOXO3 expression and activity in MCF-7 and MCF-7-EpiR cells in response to epirubicin. a The epirubicin-sensitive MCF-7 and -resistant MCF-7-EpiR cells were either left untreated or treated with 1 µM epirubicin for the times shown. Whole-cell protein lysates were then analysed by western blotting using the antibodies against 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-PERK (T981) (140 kDa), PERK (140 kDa), P-eIF2a (S51) (38 kDa), eIF2a (38 kDa), P-AKT (S473) (60 kDa), P-AKT (T308) (60 kDa), AKT (60 kDa), p27Kip1 (27 kDa) and ß-Tubulin (55 kDa) were investigated. b FOXO3, PERK, FOXM1, p27Kip1 mRNA expression after treatment with epirubicin as determined by RT-qPCR. Representative RNA expression profiles of at least three independent experiments. Three technical repeats were conducted in one experiment, and the data were normalised to L19 and displayed as means ± S.E.M. The expression trends of mRNA species between MCF-7 and MCF-7-EpiR cells are compared using two-way ANOVA (Significant ***p < 0.001, for all mRNA species, respectively)

Fig 5: Effects of silencing GCN2 on MCF-7 and MCF-7EpiRcells treated with epirubicin. A. MCF-7 and MCF-7EpiR cells were treated with 0, 5 nM, 10 nM, 25 nM, 50 nM and 100nM of epirubicin for 48 h. Fresh media were then added and maintained for 10 days before staining violet in clonogenic assays. The clonogenic results of the cells were normalised to that of the MCF-7 or MCF-7EpiR cells without GCN2 knockdown and epirubicin treatment. The clonogenic results of the cells were normalised to that of MCF-7 or MCF-7EpiR cells without epirubicin treatment (far right panel). Data are representative of 3 independent experiments. Data represent means ± SEM. Significant *P < 0.05, ***P < 0.001. (n = 3; 1-way ANOVA). Significant *P < 0.05, ***P < 0.001. B. Expression levels of signalling molecules were analysed by parallel Western blotting after transfection with siRNA in the presence of 1 µM epirubicin. Western blotting was performed to determine the protein expression levels for GCN2 (220 kDa), FOXO3 (85 kDa), P-PERK (140 kDa), PERK(140 kDa), P-eIF2a (38 kDa), eIF2a (38 kDa), P-p38 (38 kDa), p38 (38 kDa), P-JNK (54 kDa), JNK (54 kDa), p27Kip1 (27 kDa) and ß-Tubulin (55 kDa). The ratio to tubulin expression calculated the relative P-PERK, PERK and FOXO3 expression levels. C. Expression levels of GCN2, FOXO3,PERK and p27Kip1 mRNA were investigated by RT-qPCR, and the data normalised with L19 RNA levels and displayed as means ± SEM (n = 3; 2 tailed t-test). Significant *P < 0.05, **P < 0.01. Representative RNA expression profiles of one of at least 3 independent experiments are shown.