## 2 Materials and Methods
### Immunocytochemistry  
Immunofluorescence staining was used to assess the myogenic regulatory factors using specific antibodies. The myoblasts were cultivated at a density of 1 \(\times\) 10\({}^{4}\) cells per well on sterile coverslips in 24-well culture plates under the respective stress conditions. The proliferation markers were evaluated by fixing the cells after 3 h, 24 h, and 3 d in the growth medium. To examine the differentiation markers, the cells were cultivated in the myogenic differentiation medium for up to 7 d. The cells were then washed with prewarmed PBS and then fixed with 4% PFA for 20 min. After three-minute washes with PBS, the cells were permeabilized in a buffer consisting of 20 mM Hepes (Biochrom, Berlin, Germany), 300 mM sucrose, 50 mM NaCl, 3 mM MgCl\({}_{2}\) ([PERSON], Karlsruhe, Germany), and 0.5% Triton\({}^{\text{TM}}\) X-100 (pH 7, Calbiochem GmbH, Sandhausen, Germany) for 15 min. The non-specific bindings were blocked with 5% FCS diluted in PBS and supplemented with 0.05% Triton\({}^{\text{TM}}\) X-100 for 30 min. The cells were then incubated with primary antibodies diluted in the blocking buffer at 4 \({}^{\circ}\)C overnight. The primary antibodies used were anti-mouse MyoD (1:100, BD Bioscience, Heidelberg, Germany), myogenin (1:100, Santa Cruz, Biotechnology, Heidelberg, Germany), and myosin heavy chain 1 F-59 (MHC1:200, Santa Cruz, Biotechnology, Heidelberg, Germany). After five washes of 5 min each, the primary antibodies were detected by incubating the cells with Cy3-Goat anti-mouse IgG secondary antibody (diluted 1:200, Dianova, Hamburg, Germany) in the dark at room temperature for 1 h. The cell nuclei were counterstained using **4**, 6-diamidino-2-phenylindole dihydrochloride (DAPI, ThermoFisher, Waltham, MA, USA). Cells were also processed in parallel without the addition of primary antibodies, serving as negative controls. The coverslips were mounted with DABCO-Mowiol ([PERSON], Germany). The staining was examined and photographed using a fluorescence microscope equipped with the \"AxioCam MRm Rev.3\" camera and the \"AxioVision Image Analysis 4.8.2\" software. The images were quantified by using ImageJ software. To quantify the number of Myo- and myogenin-positive cells, ten random microscopic field images were photographed, then the total number of nuclei and the number of MyoD- or myogenin-positive cells were counted. The number of MyoD- and myogenin-positive cells was presented as a relative percentage of the total number of nuclei in the microscopic field. Morphometric analysis of myotubes was performed on day 7 of myogenic differentiation. For all experimental groups, ten random microscopic fields were photographed, and then MHC-positive myotubes with a minimum of two nuclei were evaluated using ImageJ. Morphometric parameters including the number, length, width, and size of the myotubes were assessed. The nuclei/cytoplasm ratio (N/C) per myotube was determined by dividing the area of the myotube by the number of nuclei for the same tube. An additional differentiation parameter, the fusion index, was obtained by dividing the number of nuclei in myotubes by the total number of nuclei in the microscopic field.