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

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — A, B Lon and ULK1 complex accumulates at ER-mitochondria contact (EMC) sites under hypoxia. HCT-15 cells treated with or without CoCl₂ (200 μM for 18 h) (A) or transfected with the plasmids encoding myc-Lon (B) were used to perform subcellular fractionation experiment. Cell lysates were analyzed by immunoblotting using the indicated antibodies. GAPDH as the loading control. Mito mitochondria, MAM mitochondria associated membranes, ER endoplasmic reticulum, cyto cytosol, PNS post nuclear supernatant. C–E Accumulation of Lon and ULK1 at the EMC/MAM was verified by confocal immunofluorescence. HCT-15 cells were transfected with the plasmids encoding Lon or treated with CoCl₂ (200 μM for 18 h) or not. The cells were fixed and immunostained by anti-Lon (green) (C), anti-ULK1 (green) (D), anti-FACL4 (ER and MAM, green) (E), anti-SERCA-2 (ER, blue), and anti-TOMM20 (mitochondria, red) antibodies. Scale bars = 10 μm. F–H Localizaton of Lon at the EMC/MAM was verified by transmission electron microscopy (TEM). HCT-15 cells were treated with CoCl₂ (200 μM for 18 h). The cells were fixed and and immunostained by anti-Lon and immunogold labeling antibodies. The immunogold electron micrographs showed Lon in (i) damaged mitochondria (M), (ii) ER around Nucleus (N), (iii) Cytosol (F) and the ER-mitochondria tethering sites (G). ER: endoplasmic reticulum. Scale bars: 100 nm. Quantitation of the percentage of ER adjacent to mitochondria in both CoCl₂ and Lon expressed HCT-15 cells and compared with control (H). The percentage was normalized by total ER and mitochondrial perimeter (n = 36 field for each condition). n = 3 biological replicates. The error bars shown in the panel represent the standard deviation from three independent experiments. ***p < 0.001.