Apoptosis analysis and caspase activity

Following transfection, HeLa cells (1×10⁵ cells/well) were plated at in 6-well plates for 48 h. Cells were stained with 10 µl Annexin V-fluorescein isothiocyanate Apoptosis Detection kit and 5 µl propidium iodide (both BD Biosciences; Franklin Lakes, NJ, USA) in darkness for 15 min at room temperature. Cell apoptosis was measured by using a flow cytometer (FACSCalibur; BD Biosciences) and analyzed using FlowJo software (version 7.6.1; FlowJo LLC, Ashland, OR, USA).

Following transfection, HeLa cells (1×10⁵ cells/well) were plated in 6-well plates for 48 h. Total protein from cells was extracted using radioimmunoprecipitation (RIPA) buffer and total protein concentration was measured using a BCA protein assay (both Beyotime Institute of Biotechnology). A total of 5 µg total protein from each sample was used to analyze caspase-3/9 activity using Caspase 3 Activity Assay kit (cat. no. C1116) and Caspase 9 Activity Assay kit (cat. no. C1158; both Beyotime Institute of Biotechnology) according to the protocol of the manufacturer. The optical density values were measured using a microplate reader (model no. 680; Bio-Rad Laboratories, Inc.) at 405 nm.