Quantifying rVvhA-Induced Cell Death

To quantify the number of rVvhA-induced cell death, cells were synchronized in the G₀/G₁ phase by culture in serum-free medium for 24 h before incubation with melatonin and rVvhA and the samples were prepared as described in previous report^{23 (link)}. rVvhA-induced cell death was detected with an Annexin V and PI staining kit (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer’s instructions. Also, to evaluate the level of mitochondrial ROS in the cells treated with rVvhA and melatonin, MitoSOX, a mitochondrial superoxide indicator, was used to detect the mitochondrial ROS level and analyzed by flow cytometry. Flow cytometry was performed by Cell Lab Quanta SC (Beckman Coulter, Brea, CA, USA) and analyzed by using flowing software 2 (developed by Perttu Terho, Turku, Finland).