Fig 2: Schematic depiction of main conclusions. Thapsigargin (Tg) binds to and blocks SERCA (the ER Ca2+ pump), thus causing ER Ca2+ depletion and ER stress. ER stress upregulates LC3B and DR5 protein levels in a manner that requires individual contributions from ATF4 and CHOP. Tg-induced activation of caspase-8 (casp8), caspase-3 (casp3), and apoptosis is strongly dependent on DR5, and partially dependent on LC3B. PERK is required for Tg-induced caspase activation and apoptosis, but not for upregulation of LC3B- and DR5 protein levels. Thus, PERK delivers an essential pro-death signal via a different pathway. The IRE1-XBP1 axis is partially required for Tg-induced upregulation of DR5, but not LC3B. Moreover, IRE1-XBP1 is required for the second-phase activation of JNK by Tg, which contributes to caspase activation and apoptosis in a cell type-dependent manner with JNK being required for Tg-induced cell death in LNCaP but not HCT116 cells

Fig 3: XBP1 activates IL-6 expression via binding to the IL-6 promoter in melanoma cells. a Sequence alignment of the putative UPR element of the Il-6 promoter from humans, rats and mice. The ACGT core is indicated in bold. b Mel-RMu cells were transfected with plasmids expressing Flag-tagged XBP1s or control (ctrl.). Chromatin immunoprecipitation (ChIP) was performed by using control IgG or anti-Flag antibody before PCR amplification of the indicated region of the IL-6 promoter. c ChIP assays of extracts from 293T cells co-transfected with the empty vector or pCMV-XBP1s plasmid together with the IL-6 or IL-6 without the ACGT core (IL-6?) promoter constructs, using anti-XBP1s antibody. d Luciferase reporter assays were performed in 293T cells cotransfected with pCMV-XBP1s plasmid together with Luc constructs under the control of the wild-type (WT) human IL-6 promoter or that with the ACGT core deleted (IL-6?). The results are from at least three independent experiments. The data are presented as the mean ± s.e.m. *P < 0.05, **P < 0.01 by two-way ANOVA

Fig 4: Breast cancer cells exhibit constitutive IRE1 RNase activity. a Expression of spliced and unspliced XBP1 mRNA was assessed in a panel of breast cancer cell lines (MCF7, SKBR3, MDA-MB-231) and the non-tumorigenic breast epithelial cell line MCF10A via RT-PCR. Tunicamycin (Tm) (1 µg ml-1)-treated MCF10A cells act as a positive control for XBP1 splicing. GAPDH was used as a loading control. b Immunoblotting of XBP1s in a panel of breast cancer cell lines (MCF7, T47D, SKBR3, MDA-MB-231, and MDA-MB-468) and the non-tumorigenic breast epithelial cell line MCF10A. Tm (1 µg ml-1)-treated MCF10A cells act as a positive control for XBP1 splicing. Actin was used as a loading control. c Q-PCR quantification of the relative mRNA levels of spliced to total XBP1 in RNA samples obtained from basal-like tumor tissue (n = 5), luminal tumor tissue (n = 4), and tumor-associated normal (TAN) tissue (n = 4). Results shown for a and b are representative of three independent experiments. *P < 0.05 based on a pairwise single factor ANOVA tests comparing each tissue type. Error bars represent s.e.m.

Fig 5: IRE1a-XBP1 axis of the UPR is selectively activated upon TLR4 stimulation.THP-1 cells were primed with either 1 µg/ml LPS alone for 24 h or 1 µg/ml LPS for 24 h followed by addition of 10 µM nigericin (NG) for 45 min. a Processing of pro-IL-1ß was analysed in cell lysates and conditioned medium by immunoblotting for full-length pro-IL-1ß and processed p17 IL-1ß. b Levels of IL-1ß were quantified in conditioned medium from untreated, LPS, LPS and NG-treated THP-1 cells by ELISA (n = 3). c Processing of caspase-1 was analysed in cell lysates and conditioned medium by immunoblotting for pro-caspase-1 and processed p10 caspase-1. d IL-6, IL-8, and TNF-a levels were quantified in conditioned medium from untreated, LPS, LPS and NG, and NG-only treated THP-1 cells by ELISA (n = 3). e UPR markers XBP1s, PERK, ATF6, phospho-eIF2a, total eIF2a, and ATF4 were analysed by immunoblotting in THP-1 post treatment with LPS alone or LPS and NG. Tunicamycin (Tm)-treated THP-1 cells served as a positive control for the UPR activation. Actin was used as a loading control. ***P < 0.001 based on a Student’s t test. Error bars represent SD.