Fig 2: IRE1α signaling is adaptive in cultured GECs subjected to proteotoxic stress.A, B Incubation with TM for 24 h stimulated the upregulation of total IRE1α, robust XBP1 splicing, and mild apoptosis (caspase-3 cleavage) in control (Ctrl) GECs. Quantification of IRE1α phosphorylation was normalized to the actin signal. IRE1α phosphorylation was absent in IRE1α KO GECs but was not affected by 4μ8C, compared to control GECs. IRE1α KO and 4μ8C-treated GECs show undetectable XBP1 splicing and greater apoptosis (immunoblots). C–F TM increased the expression of ER chaperones in control GECs. C, D Upregulation of GRP94, BiP and MANF was impaired in IRE1α KO GECs, and these results were generally recapitulated by 4μ8C. E, F The chaperone ERdj3 is a glycosylated protein. TM inhibits glycosylation and induces an underglycosylated band of ~34 kDa (both bands were used for quantification). Upregulation of ERp57 and ERdj3 was not affected by IRE1α inhibition. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. Three experiments performed in duplicate.

Fig 3: Effect of the UPR and IXA6 on FACs. (A,B) Control and IRE1α KO GECs were untreated or were incubated with adriamycin (ADR; 0.5 μM) for 24 h. Cells were then stained with anti-vinculin antibody (red; FACs), phalloidin (green; F-actin cytoskeleton) and H33342 (blue; nuclei). Representative photomicrographs (A) and quantification of FACs (B) are presented. The number of FACs was reduced after ADR, and the reduction was greater in IRE1α KO cells compared to control. Bar = 25 μm. *p < 0.05, **p < 0.01, ***p < 0.0001. Mean ± SE, 3 experiments with 10–20 measurements per group per experiment. (C and D) GECs were untreated, or were incubated with ADR together with or without IXA6 (IXA; 10 μM) for 24 h. Cells were then stained as above. IXA6 attenuated the reduction in FACs induced by ADR. **p < 0.01, ***p < 0.0001. Mean ± SE, 4 experiments with 10–21 measurements per group per experiment. (E,F) GECs were incubated with IXA6 (10 μM) for 24 h. Lysates were immunoblotted as indicated. Representative immunoblots (E) and densitometric quantification of MANF normalized to actin (F) are presented **p < 0.01. Mean ± SE, 3 experiments performed in duplicate.