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. 2021 Apr 2;22(7):3721. doi: 10.3390/ijms22073721

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© 2021 by the authors.

Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

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(A–D) Myogenic regulatory factor expression during myoblast differentiation. Quantitative analysis of the muscle-specific transcription factors MyF5 (A), MyoD (B), myogenin (C) and MRF4 (D) mRNA expression in aged myoblasts compared to controls during their differentiation processes. The mRNA expression values of MRFs in aged myoblasts have been normalized to the corresponding GAPDH mRNA and are expressed as fold changes compared to control cells. (E,F) Effects of myoblast aging on their expression of the myogenic regulatory factors MyoD and myogenin. Representative Western blots and immunoblotting quantification of MyoD (E) and myogenin (F) in aged myoblasts compared to control cells during their differentiation process. The values of the proteins of interest were normalized to each corresponding GAPDH on the same immunoblot. (G) Morphology-based analysis of control and aged C2C12 myoblasts during differentiation. Bright-field microscopy shows delayed myotubes formation in the aged myoblasts compared with the control cells over time, during myogenesis. (H) MyHC and DAPI immunostaining revealed a substantial reduction inmyotubes in aged cells. (I) Fusion index and (J) maturation index values were calculated in control and aged myotubes immunostained with MyHC. Myotubes were considered differentiated cells that contained more than three nuclei. The fusion index was defined as the percentage of nuclei present in myotubes over the total number of nuclei present in the observed field, while maturation index was defined as the percentage of nuclei present in myotubes that contained more than 10 nuclei over the total number of nuclei present in the observed field. (K,L) Morphological analysis of myotubes observed after 2 and 6 days of differentiation at both control and aged groups. Length and width of myotubes of each group have been measured and reported as mean values in the graph. Data wereselected from 10 different and randomly chosen microscopic fields. (Mean ± SE of 3 independent experiments performed in triplicate; * p < 0.05, ** p < 0.01, *** p <0.001).