Figure 4.

Elaiophylin inhibits mitophagy in C918 cells by regulating SIRT1. After pre-treatment with SRT1720 (a SIRT1 specific activator) for 24 h, C918 cells were incubated with Elaiophylin for another 24 h. (A) Western blotting was performed to analyze the proteins related to mitophagy. GAPDH and VDAC1 were used as loading controls for cytoplasm and mitochondria, respectively. (B) The membrane potential of C918 cells pre-treatment with or without SRT1720 followed by the incubation with or without Elaiophylin was evaluated by JC-1 fluorescent probe. (C) Intracellular ROS level was determined by DCFH-DA fluorescent probe. The representative images of fluorescent probe were shown on the left and the quantitative analysis of ROS level was shown on the right. (D) Colocalization of mitochondria and lysosomes. Mitochondria were stained with MitoTracker Green (200 nM), and lysosomes were stained with LAMP1 (red fluorescence). The representative images of fluorescent labeling were shown on the left and the quantitative analysis of cellular mitophagy was shown on the right (Image J was used for quantitative analysis of fluorescence co-localization, and five different visual fields of each group were selected for measurement). Data was expressed as mean ± SD of three experiments. ^** p<0.01 vs. control, ^## p<0.01 vs. Elaiophylin group.