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. 2023 Jan 24;14(1):57. doi: 10.1038/s41419-023-05599-1

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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 MCF7 cells were seeded, collected and isolated by cellular subfractionation and Percol gradient in order to separate cytoplasm (Cyto), crude mitochondria (CMito, mitochondria and other contaminating membranes), microsomes (fraction containing the ER), and mitochondria-associated membranes (MAMs, fraction containing mitochondria–ER contacts). Also kept was whole lysate (input). All fractions were prepared for the same total protein concentration and processed by Western blotting, and equal volumes of each fraction were loaded. Endogenous CPT1C was mainly localized at ER and MAMs. B MCF7 cells were transfected with human CPT1C or CPT1A tagged with mTurquoise2 (mTurq2); 48 h later, Mitotracker Orange (200 nM) was added, and incubation was 30 min. The cells were then fixed. and calnexin (used as an ER marker) was detected by immunocytochemistry. CPT1C mainly colocalized with the ER (orange spots), while CPT1A colocalized with mitochondria (light blue spots). The colocalization of CPT1C with mitochondria (the few light blue spots) may correspond to ER–mitochondria contacts. The scale bar in B is 10 µm.