VDR regulates simulated microgravity-induced atrophy in C2C12 myotubes

Figures

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  Figure 1. VDR motif is enriched in gene regions closed by simulated microgravity. (a) De novo motifs in the chromatin regions identified as regions closed by simulated microgravity in C2C12 myotubes. In the sequence logos, the height of each letter represents the relative frequency of the occurrence of the nucleotide at that position. (b) De novo motifs of the chromatin regions identified by ATAC-seq as being simulated microgravity-closed regions and as having significantly reduced H3K27Ac levels. Frequently occurring bases are represented by large letters, while infrequently occurring bases are represented by small letters.

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  Figure 2. VDR is induced in response to simulated microgravity. (a) Vdr mRNA expression in C2C12 myotubes cultured for 48 h at 1 g or under simulated microgravity (10−3 g). Data are means ± s.d. n = 5. Mann–Whitney test. *P < 0.05. (b) Western blot of VDR and β-Actin in C2C12 myotubes cultured for 48 h at 1 g or under simulated microgravity (10−3 g). Representative images of at least 3 independent experiments are shown. (c) Phase contrast images and immunocytochemical staining for VDR (green) in C2C12 myotubes cultured for 48 h at 1 g or under microgravity (10−3 g). Nuclei were counterstained with DAPI (blue). Scale bars, 50 μm.

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  Figure 3. VDR deficiency does not affect myotube differentiation. (a) Diagram showing Vdr deletion using the CRISPR-Cas9 system. (b) Western blot of VDR and β-Actin in C2C12 myotubes confirming that VDR expression was absent in Vdr KO cells. (c) Phase contrast images of control and Vdr KO C2C12 cells after differentiation for 3 or 5 days. Scale bars; 500 μm. (d) Distribution of myotube lengths. Data are means ± s.d. Mean numbers of myotubes within the indicated length ranges per 100 myotubes in control and Vdr KO C2C12 cells after differentiation for 3 or 5 days. Data are means ± s.d. Two-way ANOVA and multiple Mann–Whitney tests for multiple comparisons. ns: no significant. (e) Myod1, Myog, Myh3 and Myh4 mRNA expression in control and Vdr KO C2C12 cells after differentiation for 0, 3, or 5 days. Data are means ± s.d. Two-way ANOVA and multiple Mann–Whitney tests for multiple comparisons. ns: not significant.

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  Figure 4. VDR-deficient myotubes are resistant from simulated microgravity-induced atrophy. (a) Phase contrast images of control and Vdr KO C2C12 myotubes cultured for 48 h under control (1 g) or simulated microgravity (10−3 g) conditions. The culture conditions were identical between the two groups, except for the use of the 3D-clinostat apparatus. Scale bars: 200 μm. (b) Distribution of myotube lengths. Data are means ± s.d. Mean numbers of myotubes within the indicated length ranges per 100 myotubes in control and Vdr KO C2C12 cells at 1 g (control) or under simulated microgravity (10−3 g). Data are means ± s.d. Two-way ANOVA and multiple Mann–Whitney test for multiple comparisons. *P < 0.05. (c) Fbxo32 mRNA expression in control and Vdr KO C2C12 myotubes cultured for 48 h at 1 g or under simulated microgravity (10−3 g). Data are means ± s.d. Two-way ANOVA for multiple comparisons. *P < 0.05. (d) Western blot of FBXO32, TRIM63 and β-Actin in control and Vdr KO C2C12 myotubes cultured for 48 h at 1 g or under simulated microgravity (10−3 g). Images representative of at least 3 independent experiments are shown. Relative band intensities corresponding to FBXO32 and TRIM63 are shown in the…