Cells were treated with 0.5 × 109/mL PA or 0.25, 0.5, or 1.0 × 109/mL PA-MSHA for 24 hours, and then resuspended in serum-free media at a concentration of 1 × 106/mL. The cells were treated with 5 μL FITC-annexin V and 5 μL propidium iodide (PI; 50 μg/mL), mixed gently, and incubated at room temperature for 15 minutes in the dark. We then added 400 μL 1× Binding Buffer to each sample. Apoptosis was analyzed by flow cytometry using a FACSCalibur system (BD Biosciences, USA). For cell cycle analysis, cells were resuspended as described previously and then fixed in 5 mL of 70% ethanol at 4°C overnight. Samples were then washed twice with PBS and PI added to a final concentration of 50 μg/mL. After incubating the samples at 4°C for 30 minutes, cell cycle profiles were analyzed by flow cytometry.