Fig. 8. Lon-ROS-ULK1-FUNDC1 axis induced mitophagy benefits cell survival and tumorigenesis in vitro and in vivo.

A–C HCT-15 cells were treated with CoCl₂ (200 μM for 18 h) or not in the presence or absence of SBI-0206965 (20 μM for 24 h). The MTS assay for cell viability (A), fluorescence-based cleaved Caspase 3 apoptosis assay (B), and western blotting analysis (C) were performed. Immunoblots were obtained using the indicated antibodies. Scale bar, 100 μm (n = >50 cells/condition and 3 biological replicates). D–F HCT-15 cells were treated with CoCl₂ (200 μM for 18 h) or not in the presence or absence of Bafilomycin A1 (100 nM for 24 h). The MTS assay for cell viability (D), fluorescence-based cleaved Caspase 3 apoptosis assay (E), and Western blotting analysis (F) were performed. Immunoblots were obtained using the indicated antibodies. Scale bar, 100 μm (n = >50 cells/condition and 3 biological replicates). G Immunohistochemical analysis of ULK1, p-FUNDC1 S17, FUNDC1, LC3B, and Bcl-2 expression in Lon expressed OEC-M1 tumor generated in BALB/C Nu mice treated with or without CGP37157. Representative immunohistochemical staining of respective targets was performed using paraffin-embedded sections of tumors collected. Microscopic magnification, ×400. Scale bar, 50 μm. H Scheme of the interaction between mitochondrial Lon and ULK1 complex at the EMC/MAM promotes mitophagy under hypoxia by stabilizing FUNDC1-ULK1 complex that depends on mitochondrial Na⁺/Ca²⁺ exchanger (NCLX). Upon hypoxia, Lon promotes FUNDC1-ULK1-mediated mitophagy at the EMC/MAM site, which is dependent on the binding with mitochondrial Na⁺/Ca²⁺ exchanger (NCLX). This interaction stabilized the FUNDC1-ULK1 at the EMC and initiated the mitophagy through the regulation of Ca²⁺ levels between mitochondria and cytosol. Lon-ULK1 phosphorylates FUNDC1 at S17, and Lon-ULK1-FUNDC1 axis promotes mitophagosome-lysosome fusion. EMC ER-mitochondria contact sites, MAM mitochondria associated membranes. The scheme of this study was created with BioRender.com.