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. 2019 Jul 18;10:3187. doi: 10.1038/s41467-019-11227-4

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© The Author(s) 2019

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. 2 — Sustained mTORC1 activation impairs muscle response to denervation. a Mass variation for TA and soleus muscles in control (Ctrl) and TSCmKO (TSC) mice, after 3, 7, 14 and 28 days of denervation, compared to innervated muscle. n = 6 Ctrl, 5/4 TSCmKO (3d TA/Sol), 7/5 (7d TA/Sol) and 3 (14 and 28d) mice per genotype. b, c Fiber size distribution for type IIB (b) and IIA/X (c) fibers in TA innervated (In) and denervated (De, 28d) muscles from control and TSCmKO mice. n = 3. d Minimum mean fiber feret in TA innervated and denervated (28d) muscles from control and TSCmKO mice, distinguishing type IIB and IIA/X fibers. n = 3. e, f Immunostaining of TA control and TSCmKO muscles for MHCIIA/X (green) and laminin (red). Scale bar, 200 µm. Quantification (f) gives the proportion of type IIB fibers in TA innervated and denervated (28d) muscles. n = 3. g, h HE coloration of TA control and TSCmKO innervated and denervated (28d) muscles (6 independent muscles per group). Open arrows, arrows and arrowheads point to swollen nuclei, basophilic aggregates and vacuoles, respectively. Scale bar, 50 (g) and 10 (h) µm. i Confocal pictures of lamin (red, top) and nucleolin (red, bottom), laminin (green) and Dapi (blue) in control and TSCmKO denervated (14 and 28d) muscles (3 independent muscles per group). Arrows indicate giant, swollen myonuclei. Scale bar, 20 µm. j, k TUNEL staining in control and TSCmKO muscles after 28 days of denervation. The arrow points to a positive, swollen myonucleus, as shown in the inset with increased brightness. Scale bar, 50 µm. Quantification in (k) gives the number of TUNEL-positive nuclei detected per 100 muscle fibers in control and TSCmKO innervated muscles, and after 28 days of denervation. n = 3 mice/group. l Confocal pictures of acetylated histones H3 (Lys9; H3K9ac) and H4 (H4ac) and of trimethylated H3 (Lys4, H3K4me3) in denervated (14d) control and TSCmKO muscles (4 independent muscles per group). The arrow indicates a swollen myonucleus. Scale bar, 10 µm. Values are mean ± s.e.m.; two-tailed unpaired Student’s t-test (a) or two-way ANOVA with Tukey’s post-hoc test (d, f), *p < 0.05, **p < 0.01, ***p < 0.001. Source Data are provided in the Source Data File