Ac devd cho

For in vitro experiments, cell lines were treated with 10μM Erlotinib (AlfaAesar, Ward Hill, Massachusetts, USA), 30μg/mL Cisplatin (Mylan, Saint-Priest, France) or 0.2mL DiMethylSulfOxyde (Sigma Aldrich, Saint-Louis, Missouri, USA) for 18 hours. In order to demonstrate the specificity of the apoptotic signal using Nucview, cell lines were separated in two wells, in which were added ten μM Ac-DEVD-CHO (caspase 3 inhibitor) or DMSO for an additional 15 minutes. Cells were then harvested and a first sequence of images was acquired using the CellVizio^® system, by direct application of the optical miniprobe (Alveo-Flex AF2040, Mauna Kea Technologies) onto the cell pellets. Cells were re-suspended in 500μL of culture medium containing Erlotinib (10μM), Cisplatin (30μg/mL) or DMSO (0.2mL), and Ac-DEVD-CHO (10μM, Biotium) or DMSO. Ten minutes after addition of C3-NucView (0.2mM, Biotium), a second sequence of images was acquired using the same technique.
For flow cytometry experiments, cells were prepared and treated with Erlotinib (10μM), Cisplatin (30μg/mL) or DMSO (0.2mL), and Ac-DEVD-CHO (10μM, Biotium) or DMSO as described above. C3-NucView was added at 0.2mM. Cells were then analyzed on Cytomics FC 500 (Beckman Coulter, Fullerton, California, USA) within the hour. Five separate sets of experiments were performed.

HT and HT Bcl-2 cells were incubated with 20 µg/ml cycloheximide (CHX) (Sigma Aldrich, Brussels, Belgium) in combination with 4 µM Ac-DEVD-CHO (Biotium, CA, USA) to inhibit caspase-3. In addition, the proteasome inhibitor MG-132 (20 µM) was used for 4 h in the presence of CHX and the caspase-3 inhibitor. Samples for immunoblotting were taken at the indicated time points.