In vitro activity: Azoramide may have the protective effects of enhancing chaperone expression and reducing protein synthesis without inducing cytotoxicity and apoptosis. Azoramide may require the presence of intact IRE1 and PERK branches of the UPR to fully increase chaperone capacity. Azoramide is found to be a kind of compound with the dual property of not only boosting ER folding acutely but also activating ER chaperone capacity chronically to promote ER homeostasis. Its treatment potently protects cells against chemically-induced ER stress conditions. Azoramide preserves beta cell function and survival during metabolic ER stress. Azoramide pretreatment does not impair ER function as part of its initial action. Azoramide treatment leads to increased SERCA expression, resulting in enhanced retention of Ca+2 within the ER. Azoramide interacts with UPR pathways to promote resolution of ER stress and improve ER function.

Kinase Assay: Cells cultured under the different conditions were washed with ice-cold PBS and lysed in RIPA buffer containing a protease inhibitor cocktail (Roche, Mannheim, Germany). Protein concentrations were determined using the BCA assay kit. Total protein of 50 μg was separated on 12% SDS-polyacrylamide gels and transferred to nitrocellulose membrane, and the membrane was blocked with 5% nonfat milk in Tris-buffered saline (TBS). The membrane was then incubated with the appropriate primary antibodies in TBS at 4 °C overnight. Antibodies for Runx2, PPARγ, Fabp4, β-catenin, and phospho-β-catenin were from Cell Signaling Technology (Beverly, MA, USA). GLP-1R antibody was from Ruiying Biological, and antibodies against PKAc and β-actin were from Sangon. Secondary antibodies conjugated to IRDye 800 were detected using an Odyssey infrared imaging system. All Western blots were independently replicated at least three times and the intensities of the bands were quantified using ImageJ software.

Cell Assay: C3H10T1/2 cells were seeded on coverslips. After different treatment, cells were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.1% Triton X-100 in TBS for 15 min. After being blocked in 5% FBS in PBS for 30 min, cells were incubated with anti-β-catenin (Cell Signaling Technology) overnight at 4 °C. After washing three times with PBS, cells were incubated with DyLight(TM) 594-conjugated secondary antibodies (Thermo Scientific) for 60 min at room temperature and then stained with 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich) for 10 min. Slides were then washed three times and mounted. Immunofluorescence was detected using an Olympus inverted fluorescence microscope.