Fig 2: IRE1a-XBP1 pathway promotes melanoma cell proliferation by regulating IL-6/STAT3 signaling. a Immunoblotting analysis of the indicated proteins and b XBP1 splicing levels, as determined by real-time PCR in Mel-RMu cells transfected with plasmids pCMV-IRE1a (IRE1) or pCMV (ctrl.) and treated with anti-IL-6 antibodies (anti-IL-6). c Immunoblotting analysis of the indicated proteins in Mel-RMu cells transfected with plasmids pCMV-XBP1 (XBP1s) or pCMV (ctrl.) and treated with anti-IL-6 antibodies (anti-IL-6). d, e Cell proliferation was analyzed by CCK8 assay (d) and BrdU assay (e) in Mel-RMu cells transfected with plasmids pCMV-IRE1a (IRE1) or pCMV (ctrl.) and treated with anti-IL-6 antibodies (anti-IL-6). f, g Cell proliferation analyzed by CCK8 assay (f) and BrdU assay (g) in Mel-RMu cells transfected with plasmids pCMV-XBP1 (IRE1) or pCMV (ctrl.) and treated with anti-IL-6 antibodies (anti-IL-6). The results are from more than three independent experiments. The data are presented as the mean ± s.e.m. *P < 0.05, **P < 0.01 by Student’s t test, one-way or two-way ANOVA relative to the ctrl. group; # P < 0.05 by Student’s t-test relative to IRE1 + anti-IL6 or XBP1s + anti-IL6 groups, as indicated

Fig 3: IRE1 and XBP1 are partially required for Tg-induced caspase activation and upregulation of DR5-, but not LC3B expression in LNCaP cells. a LNCaP cells were transfected for 2 d with the indicated siRNAs (siCtrl = non-targeting control siRNA), employing two different siRNA oligoes for each target (designated by - 1 and - 2). After 30 h of treatment with 0.02% DMSO (“DMSO”, also transfected with siCtrl) or 100 nM Tg in the absence of presence of 0.5 µM JNK inhibitor JNK-IN-8 (JNKi, also transfected with siCtrl), whole cell lysates were prepared and subjected to western blotting with the indicated antibodies; Casp3 = caspase-3 (only cleaved caspase-3 bands are shown), cl-PARP = cleaved PARP, p-JNK = phospho-JNK. The positions of molecular weight markers are indicated to the left. In the presence of JNK-IN-8, the two p-JNK bands shift towards slightly slower migration due to covalent binding of the inhibitor to the JNK1 and JNK2 isoforms. One representative blot out of at least 3 independent experiments. b-g Quantifications of western blots from (a), normalized to the tubulin loading control and then to the siCtrl+Tg condition. Mean ± SEM from 4 (b-f) or 3 (g) independent experiments. Dots represent individual values, with a separate color for each experiment. *P < 0.05, **P < 0.01, ***P < 0.001, ns; not significant, One-way ANOVA compared to the Tg + siCtrl condition

Fig 4: Addition of MKC8866 does not impact upon LPS-mediated increases in CASP1, IL1ß, or NLRP3 transcript.THP-1 cells were treated with 1 µg/ml LPS in the presence or absence of 20 µM MKC8866 for the indicated times after which RNA was extracted. a Q-PCR was carried out to assess relative expression of IL1ß and NLRP3 and CASP1 transcripts (n = 3). Error bars represent SD. b RT-PCR was carried out to assess XBP1 splicing and GAPDH mRNA expression.

Fig 5: DGAT2 is an IRE1a RIDD substrate.a–d MDA-MB-231 cells were either a, b transfected with non-coding (nc) and IRE1 siRNA for 72 h (n = 3 biologically independent experiments), c treated with Tunicamycin (Tm, 5 µg/ml), Thapsigargin (Tg, 0.25 µM), Brefeldin A (BFA, 0.5 µg/ml), MKC8866 (20 µM) for 24 h (n = 4 biologically independent experiments) or d pre-treated with actinomycin D (2 µg/ml) for 2 h followed by incubation of cells with Tg (0.25 µM) and MKC8866 (20 µM) for 8 h (n = 3 biologically independent experiments). e MDA-MB-231 XBP1 knockout (KO) cells were treated with DMSO or 20 µM MKC8866 in the presence or absence of 1 µM Tg (n = 3 biologically independent experiments). RT-qPCR quantification of DGAT2 transcript levels relative to a, c, d MRPLP19 and e ACTB and normalized to control at 0 h time point. b Immunoblotting of total IRE1a, XBP1s and ACTIN. f–h Expression of DGAT2 relative to MRPL19 in f HCC1806 (n = 4 biologically independent experiments), g MDA-MB-468 (n = 5 biologically independent experiments) and h BT-549 cells (n = 3 biologically independent experiments) treated with DMSO or 20 µM MKC8866 in the presence or absence of 1 µM Tg for 24 h. i, j RNAfold prediction of secondary structure of mRNA fragments of i DGAT2 and j XBP1 mRNA. IRE1a consensus cleavage sequences are highlighted in red. k, l In vitro transcribed k wild type (WT) DGAT2, c.260G>A DGAT2 mutant (MUT DGAT2)and l XBP1 mRNAs were incubated with recombinant hIRE1a in the presence or absence of 20 µM MKC8866. Arrows indicate cleavage products (n = 4 biologically independent experiments). m MDA-MB-231 cells stably expressing empty vector (EV), FLAG-tagged wild type DGAT2 (WT DGAT2) or guanine 260 to adenine mutated FLAG-tagged DGAT2 (MUT DGAT2) were treated with 20 µM MKC8866 for indicated time points. Representative immunoblots of FLAG and ACTIN for three biologically independent experiments. Indicated p values, based on a comparison of the two groups using an unpaired two-tailed t test or c–h one-way ANOVA with Bonferroni’s multiple comparisons post hoc tests. Values with p < 0.05 are considered statistically significant. Data are presented as mean values ± s.d. Source data are provided as a Source Data file.