Figure 5
From: Rbm24 displays dynamic functions required for myogenic differentiation during muscle regeneration
Rbm24 silencing affects skeletal muscle regeneration. (a) Muscle injury was induced by CTX injection in the tibialis anterior. Satellite cells previously transfected with GFP reporter and shScramble or shRbm24 at a 1:10 ratio were implanted in the injured area one day after injury. Regenerating muscles were collected at indicated time points. (b) Images illustrating transplanted satellite cells in the tibialis anterior 3 days after injury. (c) Analysis by qRT-PCR of myogenin mRNA expression. Rbm24 knockdown affects the increase of myogenin mRNA expression in regenerating muscle tissues 3 and 6 days after injury. Data are the mean ± s.e.m. from three independent experiments. Two-way ANOVA shows a significant effect of both variables, i.e. time and shRbm24, in the presence of a significant interaction. ANOVA for treatment F (df 1.8) = 8.370; time F (1.808, 8.679) = 11.41; interaction F (5.24) = 6.108, p < 0.05; *p < 0.05 by Tukey’s HSD test. (d) RT-PCR analysis of coro6 muscle-specific alternative splicing. Rbm24 knockdown affects the inclusion of muscle-specific exon (black box) throughout the regeneration period. Full-length gel is presented in Supplementary Fig. S4. (e) Representative images of GFP-positive newly formed myofibers. Equal numbers of satellite cells (2 × 105) were transplanted into CTX-injured tibialis anterior muscles. At different time points after injury, satellite cells transfected with shRbm24 produce less newly formed myofibers, compared to the control. DAPI was used to stain nuclei. Scale bar: 20 µm. (f) Quantification of newly formed myofibers at indicated time points after muscle injury. The number of GFP-positive cells in shScramble-transfected conditions is set to 1 as a reference. Data are the mean ± s.e.m. from three independent experiments. Two-way ANOVA shows a significant effect of shRbm24 independently of time. ANOVA for treatment F (df 1.14) = 18.28; *p < 0.05 by Tukey’s HSD test. (g) Quantification of GFP-positive newly formed myofibers containing at least two centralized nuclei at 6 days of regeneration. More than 100 regenerating myofibers were analyzed in each condition. Data are the mean ± s.e.m. from three independent experiments. *p < 0.05 by Student’s t test.
