Phalloidin atto 633

The differentiation status of the primary muscle cells was evaluated after fluorescence staining assay, with the confocal microscope (Olympus FV1000, Tokyo, Japan). After 5 days of culture, cells were fixed in 4% paraformaldehyde in PBS (Life Technologies, Houston, USA; 10 min, RT), and washed with ice-cold PBS. Next, the cells on slides were permeabilised in Triton X-100 (Sigma-Aldrich, St Louis, USA) and washed with PBS (Sigma-Aldrich, St Louis, USA). Finally, cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific, Waltham, USA) and actin filaments with phalloidin-Atto 633 (Sigma-Aldrich, St Louis, USA). After staining, the cells were washed with PBS, and slides were mounted under Fluoromount-G medium (Southern Biotech, Birmingham, USA). On the stained slides, the fusion index was calculated as a ratio of nuclei in the multinucleated myotubes (more than 2 myonuclei) to the total number of nuclei (mononucleated and multinucleated) [51 (link), 52 (link)] in the randomly selected visual fields (n = 8). Next, the relative to control fusion index was calculated from the obtained results, as a ratio of the fusion index, for each group, compared to the control results. The nuclei were counted manually.

The changes in the structure of the cell skeleton and nucleus were evaluated by confocal microscopy (Olympus FV1000, Tokyo, Japan). Cells were grown on a coverslip in a 6-well plate for 4 days and then the test reagents were added. After 24 h of incubation with MEL, complex cells were fixed with 4% paraformaldehyde in PBS (Life Technologies, Texas, TX, USA) for 10 min at room temperature and washed with ice-cold PBS. The next step was incubation in Triton X-100 (Sigma-Aldrich, Missouri, MO, USA) and washing with PBS (Sigma-Aldrich, Missouri, MO, USA). Then, the cells were incubated with normal goat serum as a blocking solution (Chemicon International, California, CA, USA). Actin filaments were stained with phalloidin-Atto 633 (Sigma-Aldrich, Missouri, MO, USA) and cell nuclei with 4′,6-diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific, Massachusetts, MA, USA). Each group was prepared in 5 replications. The photos were taken in 7 randomly selected fragments.

The differentiation of cells was evaluated using a confocal microscope (Olympus FV1000, Tokyo, Japan). For imaging, cells were fixed after 5 days of culture using 4% paraformaldehyde in PBS (Life Technologies, Texas, TX, USA) for 10 min at room temperature and washed with ice-cold PBS. Then cells were incubated in Triton X-100 (Sigma-Aldrich, Missouri, MO, USA) and washed with PBS (Sigma-Aldrich, Missouri, MO, USA). Subsequently, cells were incubated with blocking solution, i.e., normal goat serum (Chemicon International, California, CA, USA). Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific, Massachusetts, MA, USA) and actin filaments with phalloidin-Atto 633 (Sigma-Aldrich, Missouri, MO USA). After staining, the cells were washed with PBS. The fusion index was calculated for the control group and cells with CEME supplementation from randomly selected visual fields (n = 7) and presented as the number of nuclei in the multinucleated myotubes divided by the total number of nuclei. The nuclei were counted manually.

All immunofluorescence experiments were performed on cells seeded on glass in MatTek plates. Cells were fixed in 4% paraformaldehyde, washed with phosphate-buffered saline (PBS) containing 0.01% Triton X-100, and permeabilised by incubation in PBS containing 0.2% Triton X-100 for 20 min at room temperature. Samples were subsequently blocked for 1 h with PBS containing 5% BSA and 0.01% Triton X-100 before incubation with Phalloidin–Atto633 (Sigma; 68825-10NMOL) and DAPI (Sigma; d9542-5 mg) in PBS containing 5% BSA and 0.01% Triton X-100 for 1 h at room temperature. Primary antibodies used were anti-YAP1 (D8H1X; 14074; Cell Signaling Technology; 1:500), and anti-Flag (F1804; Sigma; 1:100), and were incubated overnight at 4°C. Primary antibody was washed off in three washes of 5 min using PBS containing 0.01% Triton X-100. Secondary antibodies used were donkey anti-rabbit IgG Alexa Fluor 488 (Thermo Fisher Scientific; A21206; 1:200) and donkey anti-mouse IgG Alexa Fluor 555 (Thermo Fisher Scientific; A31570; 1:200). Samples were retained in PBS until imaging.

The formation of the actin ring was visualized on Days 6 and 15. The cells were fixed with 2% paraformaldehyde in PBS for 20 min. Thereafter, 0.5% Triton-X-100 was incubated with the cells for 2 min, with subsequent thorough PBS washing. Phalloidin-Atto 633 (10 nM, Sigma Aldrich, Steinheim, Germany) 1:500 was added for 45 min. Cell nuclei were visualized using Hoechst 34580 (10 ng mL⁻¹, Invitrogen, Carlsbad, CA, USA), diluted 1:1500 for 15 min. The cells were visualized using an epifluorescence microscope (Olympus U-RFL-T, IX-51, Olympus, Tokyo, Japan).