Figure 2
Role of calpain and PKC in ROS generation and cell death induced by silibinin. (A) Effect of inhibitors of calpain and PKC on silibinin-induced ROS generation. Cells were exposed to 30 μM silibinin in the presence or absence of 0.5 μM calpain inhibitor (CHO), 1 μM GF 109203X (GF), 1 μM rottlerin (Ro), and 800 units/ml catalase (Cat) and ROS generation was estimated by measuring changes in DCF fluorescence using FACS analysis. Data are mean ± SEM of five independent experiments performed in duplicate. *p < 0.05 compared with silibinin alone. (B) Effect of PKC inhibitors on silibinin-induced cell death. Cells were exposed to 30 μM silibinin in the presence or absence of 1 μM GF 109203X (GF) and 1 μM rottlerin (Ro) and cell viability was measured by MTT assay. Data are mean ± SEM of four independent experiments performed in duplicate. *p < 0.05 compared with silibinin alone. (C) Effect of silibinin on PKCδ activation. Cells were exposed to 30 μM silibinin for various times and PKCδ phosphorylation was estimated by Western blot analysis. (D) Effect of calpain inhibitor on PKCδ phosphorylation. Cells were exposed to 30 μM silibinin for 10 min in the presence or absence of 0.5 μM calpain inhibitor (CHO) and PKCδ phosphorylation was estimated by Western blot analysis.