Q vd oph qvd

SUDHL4 (ATCC, CRL-2957), JeKo-1 (ATCC, CRL-3006) and RAMOS (ATCC, CRL-1596) cells were cultured in R10 medium (RPMI 1640 [Gibco] with 1% penicillin-streptomycin [Life Technologies] and 10% heat inactivated fetal bovine serum [FBS; Sigma]). Cells were treated with 50, 500 or 2000nM selinexor (KPT-330, provided by Karyopharm Therapeutics), 50nM leptomycin B (Sigma) or DMSO control (for the indicated 0nM negative controls) for 16 hours at 37°C before use. To prevent drug-induced apoptosis, cell lines were incubated with Q-VD-OPh (QVD) (10-20µM) (Sigma) for 30 minutes prior to addition of XPO1 inhibitors or DMSO control.

Since APPΔC31 is typically produced at very low levels and below the level of detection in the in vitro systems used here, it was necessary to induce this caspase cleavage in order to then knock it down. We previously reported the ability of statins, including simvastatin and cerivastatin, to stimulate the caspase cleavage of APP (Descamps et al., 2011 (link)). Here, we utilized this ability to discover APP C-terminal caspase cleavage inhibitors using CHO-7W cells. Cells were treated with activated simvastatin at 6 doses ranging from 100 nM to 10 μM, for 7.5 and 24 h, at which time APPΔC31 was measured in cell lysates using the AlphaLISA. Simvastatin was activated by opening the lactone ring before use in cell culture as previously described Dong et al. (2009) (link); 8 mg simvastatin was dissolved in 0.2 mL of 100% ethanol (0.019 mM), with subsequent addition of 0.3 mL of 0.1 N NaOH and the solution was heated at 50°C for 2 h and then neutralized with HCl to pH 7.2. The resulting solution was brought to 1 mL final with distilled water, and aliquots were stored at -80°C.
In a separate experiment, CHO-7W cells were treated with: simvastatin, cerivastatin, and atorvastatin at 1, 5, and 10 μM, 5 μM simvastatin plus 30 μM pan-caspase inhibitor Q-VD-OPh (QVD, Sigma-Aldrich), and no statins, all for 24 h. APPΔC31 was determined in cell lysates by ELISA.

To assess protein stability, cells were treated with 50 μg/ml cycloheximide (Sigma) following siRNA transfection for 72 h. At the given timepoints, cells were washed in ice‐cold PBS and prepared for SDS‐polyacrylamide gel electrophoresis (SDS‐PAGE) as below. For inhibition of apoptosis, siRNA transfected cells were treated with 10 μM caspase inhibitor Q‐VD‐OPh (QVD, Sigma) 24 h after knockdown.

The FACSCanto™ II flow cytometer was used for the experiments and FACS Diva software for analyses.
BT474, BT474-RH, and BT474-TDM1R cells (100,000 cells/well in p6 multiwell) were collected after 24 h of treatment and fixed in ethanol (70%, ice-cold, 30 min). Cell pellets were washed in PBS with 2% BSA and incubated in the dark (1 h, 4 °C) with Propidium iodide/RNAse staining solution (Immunostep, Salamanca, Spain) to analyze the cell cycle.
BT474, BT474-RH, and BT474-TDM1R cells (100,000 cells/well in p6 multiwell) were collected after 72 h of treatment and stained for 1 h with Annexin V Binding Buffer containing Annexin V-DT-634 (AV) (Immunostep) and Propidium iodide (PI) (2 mg/mL) to analyze the cell death. Broad-spectrum inhibitor Q-VD-Oph (QVD) (10 μM, Sigma Aldrich) was added 45 min previous drug exposure.