Enzastaurin-induced accumulation of β-catenin triggers ER stress response in tumor cells. (A) Differentially regulated genes of the ER stress pathway in MM cells treated with enzastaurin vs control were delineated by oligonucleotide microarray analysis (human U133A 2.0 Affymetrix GeneChip), followed by data analysis and modified graphical display using the Ingenuity Pathway software (for further information, see also www.ingenuity.com). Analysis with Ingenuity Systems was performed under the institutional license of Dana-Faber Cancer Institute. Gene overexpression is shown in pink, down-regulation in green. (B,C) Protein profiling of MM cells exposed to enzastaurin. MM.1S cells were exposed to enzastaurin for increasing time periods (B) or concentrations (C), followed by immunoblot analysis with the indicated antibodies. Tunicamycin (TM) was used as a positive control for ER stress signaling. (D) Knockdown of β-catenin protects against enzastaurin-induced growth inhibition. MM.1S cells were transiently transfected with mock or β-catenin siRNA and treated with enzastaurin (5 μM, 6 hours), followed by immunoblotting with the indicated antibodies (top) or proliferation assay ([³H]dT uptake, bottom). [³H]dT uptake was measured during the last 5 hours of treatment. Columns represent mean of experiments done in triplicate; bars represent SD. (E) Knockdown of CHOP/GADD153 partly attenuates enzastaurin-induced growth arrest. MM.1S cells were transiently transfected with mock or CHOP/GADD153 siRNA, treated with enzastaurin (2.5 μM), and immunoblotted with indicated antibodies (top) or assayed for ³H[dT] (bottom). (F) Indicated cells were treated with enzastaurin or BIM I (both 5 μM, 4 hours), followed by immunoblotting with the indicated antibodies. (G) Primary MM cells were treated with the indicated concentrations of enzastaurin (8 hours), followed by immunoblotting with the indicated antibodies.