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. 2023 Mar 16;14(3):199. doi: 10.1038/s41419-023-05723-1

Fig. 7. Lon binds with mitochondrial Na+/Ca2+ exchanger to promote FUNDC1-ULK-mediated mitophagy in the EMC/MAM site.

Fig. 7

A, B Ca2+mito (mt-lar-GECO) and Ca2+cyto (Fura-2 AM) were measured by live-cell microscopy in OEC-M1 treated with CoCl2 (300 µM-18h) or transfected with Lon in presence or absence of CGP37157 (10 µM-4h). ATP (100 µM) was used as an agonist and further measured and analyzed (n = 3). C OEC-M1 cells were treated with CoCl2 (300 µM-18 h) in the presence or absence of CGP37157 (10 µM-4 h). Cell lysates were analyzed by immunoblotting using the indicated antibodies. GAPDH as the loading control. D OEC-M1 cells were transfected with Vector, Lon, NCLX (48 h) in the presence or absence of CGP37157 (10 µM-4h) and shNCLX transfected OEC-M1 cells were co-transfected with NCLX and Lon plasmids and incubated for 48 h. Cell lysates were analyzed by immunoblotting using the indicated antibodies. GAPDH as the loading control. E, F Lon and ULK1 complex accumulation at EMC/MAM sites were abolished upon NCLX inhibition under hypoxia. OEC-M1 cells transfected with the plasmids encoding myc-Lon (E) or treated with CoCl2 (200 μM for 18 h) (F) in presence or absence of CGP37157 (10 µM-8h) were used to perform subcellular fractionation experiments. Cell lysates were analyzed by immunoblotting using the indicated antibodies. GAPDH as the loading control. Mito mitochondria, EMC ER-mitochondria contact sites, MAM mitochondria associated membranes, WCL whole cell lysate.