In vitro activity: In Bcap-37 cells, Spautin-1 dramatically enhanced cell death in glucose-free media and induces apoptotic morphology. In Bax-Bak DKO cells, spautin-1 inhibits etoposide induced autophagic cell death. Spautin-1 promots the degradation of Vps34 complexes by regulating the deubiquitination activity of USP10 and USP13, and reduces the levels of PtdIns3P. In PDGF-treated cells, spautin-1 stabilizes α-smooth muscle cell actin and calponin, prevents actin filament disorganization, diminishes production of extracellular matrix, and abrogates VSMC hyperproliferation and migration. In CML cells, spautin-1 markedly inhibits IM-induced autophagy by downregulating Beclin-1, and enhances IM-induced apoptosis by inactivating PI3K/AKT and activating downstream GSK3β. Spautin-1 also specifically reduces infectious dengue virus titers in BHK-21 cells.

Kinase Assay: Apoptotic morphology was studied by staining the cells with Hoechst 33258 (KeyGen Biotech) fluorescent stain. Cells (1×105/ml) were seeded into a 12-well plate with indicated concentration of IM (500 nM) for 12 h. Then spautin-1 (10 μM) or DMSO was added to K562 medium for further 36 h. After incubation, cells were stained with 20 mg/ml of Hoechst 33258 for 10 min and observed under a fluorescence microscope (Olympus).

Cell Assay: Spautin-1 promotes the degradation of Vps34 PI3 kinase complexes via inhibiting two ubiquitinspecific peptidases, USP10 and USP13, that target the Beclin1 subunit of Vps34 complexes. Spautin-1 had no effect on the growth and survival of Bcap-37 cells under normal culture conditions but dramatically enhanced cell death in glucose-free media. Under glucose-free conditions, western blotting for LC3 further confirmed that autophagy was induced, which was inhibited by spautin-1. Similar results were obtained with MCF-7 and BT549 cells. Therefore, spautin-1 can sensitize tumor cells to apoptosis under nutritional deprived conditions.