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. 2022 Nov 11;13:6869. doi: 10.1038/s41467-022-34694-8

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© The Author(s) 2022

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. 5 — a Graph shows the kinetics of fracture incidence and skeletal muscle weight of Ecsit^Prx1 and Ecsit^fl/fl mice (n = 5, left). Representative macroscopic images of the gastrocnemius (GA) muscle at the age of P0 and P10 (right). b Quantification of skeletal muscle weight (left) and a ratio of skeletal muscle weight to body weight (right) are displayed (n = 5). c, d P0 (c) or P15 (d) Ecsit^Prx1 and Ecsit^fl/fl tibialis anterior (TA) muscles were stained for H&E or immunostained for MF20, COL1α1, or ECSIT. e mRNA levels of myogenic genes in P10 Ecsit^Prx1 and Ecsit^fl/fl TA muscles (n = 16). f Total cell number (n = 5) and flow cytometry analysis showing the frequency of muscle satellite cells (MuSCs) and skeletal muscle resident stem/progenitor cells (Ecsit^fl/fl, n = 9, Ecsit^Prx1, n = 5) in P10 Ecsit^Prx1 and Ecsit^fl/fl GA muscles. Supplemental Fig. 11 demonstrates the gating strategy for this analysis. g GFP-expressing Prx1⁺ skeletal cells in P3 Prx1-cre;Rosa26^mTmG GA muscle. Red: Prx1^- bone marrow and muscle. h, i Tibialis anterior muscles or femurs of E21 Ecsit^Myf5 and Ecsit^fl/fl embryos were stained for H&E, Von Kossa, or immunostained for MF20 (h). mRNA levels of Ecsit and myoblast genes in the skeletal muscle (i, Ecsit^fl/fl, n = 4−5; Ecsit^Myf5, n = 3). j Human 17Ubic myoblasts expressing control (shCtrl) or ECSIT shRNA (shECSIT) were cultured under myogenic conditions. 6 days later, expression of MF20 (green) and myogenic genes was assessed (n = 4). k, l Protein levels of NDUFAF1, ECSIT and NDUFS3 (k, left) or the ratio of mtDNA (Ndufv1) to nDNA (18s, k, right) in shCtrl or shECSIT-expressing 17Ubic cells. Fluorescence microscopy shows MitoTracker- and TMRE-stained cells and relative quantification of deep red signal intensity (n = 6, l). A two-tailed unpaired Student’s t-test for comparing two groups (b, e, f, i, j, k, l; error bars, data represent mean ± SD). Data are representative of three independent experiments. Scale bars = c, d, 60 μm; g, 75 μm; h, 30 μm; j, 100 μm; l, 10 μm.