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. 2024 Apr 1;56(4):922–934. doi: 10.1038/s12276-024-01199-x

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© The Author(s) 2024, corrected publication 2024

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — a Immunoblotting of whole-cell lysates from myoblasts and myotubes differentiated for 1, 3, and 5 days. b qPCR analysis of Crls1 expression in myoblasts and myotubes differentiated for 1, 3, and 5 days (n = 3). c Live-cell staining of cardiolipin and mitochondria by NAO and TMRM in myoblasts and myotubes differentiated for 1–5 days. Scale bar: 50 μm. d Immunoblotting of whole-cell lysates from scramble- and shCrls1-transfected C2C12 cells. e CRLS1 protein expression relative to tubulin (n = 3). f qPCR analysis of Crls1 gene expression in the Crls1-knockdown cell line (n = 6). g qPCR analysis of Crls1 gene expression in the Crls1-knockdown cell line during myogenesis (n = 3). h Total cardiolipin levels in myoblasts and myotubes differentiated for 1, 3, and 5 days (n = 4). i Representative images of myosin heavy chain (MyHC) staining of C2C12-derived myotubes at myogenic differentiation Day 5 after infection with scramble or shCrls1. 4′,6-Diamidino-2-phenylindole (DAPI) was used as a nuclear counterstain. Scale bar: 100 μm. j Measurement of the green fluorescence intensity in Myhc-stained myotubes. (n = 4). k Representative images of myosin heavy chain (MyHC) staining of C2C12-derived myotubes at myogenic differentiation Day 5 after infection with scramble or shCrls1. Scale bar: 100 μm in length. l Measurement of the green fluorescence intensity in Myhc-stained myotubes. (n = 3). m MitoTracker green staining of Crls1-mCherry-transfected C2C12 cells. Blue: Hoechst 33342, red: mCherry-Crls1, green: MitoTracker-green FM, yellow: merged. (right) Enlarged view of the boxed areas. Scale bar: 50 μm. The data are presented as the mean ± SD of n ≥ 3 independent experiments. P values were calculated using an unpaired Student’s t test. *P < 0.05; **P < 0.01; ***P < 0.001; paired t test (e and f). n.s.: nonsignificant. scramble: untargeted shRNA-Lentivirus-infected cell line; shCrls1: Crls1-targeting shRNA-lentivirus-infected cell line.