Identification of an Ire1α endonuclease inhibitor. (A) Chemical structure of inhibitor STF-083010. (B) STF-083010 inhibited endogenous XBP1 mRNA splicing. RPMI 8226 cells were treated with 300nM thapsigargin (Th), 60μM STF-083010, or both for the indicated amount of time, and the relative XBP1 splicing was determined by reverse-transcribed polymerase chain reaction. Solid arrow indicates the unspliced form; and broken arrow, the spliced form. (C) STF-083010 inhibits the production of sXBP1 protein but not the autophosphorylation of Ire1α. The indicated MM cell lines were treated for the indicated times with 300nM Th and 60μM STF-083010, and sXBP1 protein was detected by immunoblotting (left panel). RPMI 8226 cells were treated with 300nM Th and 60μM STF-083010 for the indicated times, and the levels of phosphorylated and total Ire1α were detected using specific antibodies (right panel). (D) STF-083010 effect on cell-free Ire1α RNase (endonuclease) activity. Upper panel: hIre1 was incubated with uniformly labeled (³²P) HAC1 508-nt transcript for 30 minutes in the presence of increasing concentrations of STF-083010 (1-100μM). HAC1 mRNA cleavage reaction was analyzed by separation of products on denaturing polyacrylamide gels, followed by autoradiography. Lower panel: Quantitation of HAC1 mRNA processing showing half-maximal inhibition at approximately 25μM. Error bars represent SEM of 3 independent experiments. (E) Effect of STF-083010 on cell-free Ire1α kinase activity. Upper panel: hIre1α was incubated with ³²P-γATP and increasing concentrations (0-100μM) of STF-083010. Ire1α autophosphorylation was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by autoradiography to determine the amount of ³²P incorporation (³²P-hIRE1). Lower panel: Kinase activity showed no significant change during coincubation with STF-083010. Error bars represent SEM of 3 independent experiments.