Mito-Kaede photoactivation and chase experiment for mitophagy: mitophagy flux response toward various stimulations

Mitophagy, a crucial mitochondrial quality control system for cellular stress adaptation, is a key focus in pathophysiology and drug discovery. Developing a simple and versatile mitophagy flux assay is vital for advancing our understanding of cellular responses. Addressing a gap in systematic methods, we employ the photoactivatable fluorescent protein mito-Kaede in C2C12 myocytes, demonstrating its remarkable versatility in quantifying mitophagy flux responses under various stimuli, including carbonyl cyanide m-chlorophenyl hydrazone (CCCP), TNF-α, lipopolysaccharide (LPS), and hypoxia. This study underscores the validity and distinctive advantages of the mito-Kaede assay through comparative analysis with conventional assays including Western blotting (WB), potentially providing valuable insights for both mitophagy flux analysis and drug development.