Figure 4. Puerarin prevents mitochondrial autophagy by inhibiting dephosphorylation of FUNDC1. PH0321, a protein phosphatase inhibitor, treated 20% CSE-induced HBECs and then co-cultured with puerarin at the concentration of 200 μg/mL. (A) The protein expression of FUNDC1 and p-FUNDC1 in CSE-induced HBECs were analyzed by Western blotting. (B) Flow cytometry (JC-1) was used to detect changes in mitochondrial membrane potential in CSE-induced HBECs. (C) Mitochondrial ROS levels were detected by flow cytometry (DCFH-DA). (D) The content of ATP in CSE-induced HBECs was detected with kits. (E) Western blotting was used to detect the expression of mitochondrial autophagy-related proteins such as PINK1 and Parkin. β-actin was used as an invariant internal control for calculating protein-fold changes. N=6, ** P<0.01.