Pan caspase inhibitor z vad fmk

Cell apoptosis was analyzed at 48 hours after drug treatment [11 (link)]. Pro-survival protein Bcl-2 was determined by Western blot. A measurement of the extent of apoptosis was carried out using an annexin V detection kit (Invitrogen, ThermoFisher, Waltham, MA, USA) to label cell surface phosphatidylserine of apoptotic cells. Briefly, treated cells were trypsinized and washed twice with phosphate-buffered saline, then suspended in binding buffer (10 mM HEPES, pH 7.4, 140 mM NaCl and 2.5 mM CaCl₂). To each 100 μl cell suspension was added 5 μl annexin V conjugated to fluorescein isothiocyanate (FITC), cells were incubated at room temperature for 30 min. Stained cells were analyzed immediately by flow cytometry. To further monitor the status of cell apoptosis, T-DM1 treated cells were live stained with annexin V and propodium iodide (PI) together, co-staining images were taken by inverted fluorescence microscope. Annexin V stained cells with PI positive staining were excluded as necrotic cells, apoptotic cells with only annexin V staining were counted and divided by total cells as the percentage of apoptosis. Pan-caspase inhibitor Z-VAD-FMK (Enzo, Farmingdale, NY, USA) with working concentration 20 μM was introduced together with T-DM1 for rescue experiment, cell viability was determined as described in cell viability assay.

Specific antibodies to p53, HSP70, HSP60, HSP90β, β-tubulin, p21, GAPDH, and HSP90β siRNAs were purchased from Santa Cruz (Santa Cruz, CA, USA). Polyclonal antibodies to Bax, Bcl-2, cytochrome c, and β-actin were obtained from Millipore (Temecula, CA, USA). Polyclonal antibodies to caspase-3, -7, -9, PARP, CDK2, -4, -6, cyclin D, cyclin E, N-cadherin, E-cadherin, claudin, AKT, and phospho-AKT^Ser473 were purchased from Cell Signaling (Beverly, MA, USA). Pan-caspase inhibitor (Z-VAD-FMK) was obtained from Enzo life science (New York, NY, USA).
The overall structure flowchart is shown in Figure 6 as follows: