High glucose and l glutamine

For live-cell imaging, 30,000 cells were seeded on laminin-rich coated 3.5 cm glass-bottom dishes (WPI, Sarasota, FL, USA, cat. no. FD35-100). One hour after seeding, the medium was exchanged to FCS-free DMEM (high glucose and L-Glutamine, Gibco, Paisley, United Kingdom) supplemented with Penicillin-Streptomycin. The cells were incubated for an additional 23 h. Prior to imaging, the medium was exchanged for DMEM medium without phenol red and FCS (FluoroBrite DMEM, Gibco, Paisley, United Kingdom). Live-cell imaging was performed using a spinning disc microscope (Zeiss Axio Observer Z1, inverse, fully motorized, with hardware autofocus, Yokogawa CSU-X1-A1 Nipkow spinning disc unit) and a Plan-Apochromat 63×/1.4 DIC Oil objective. For UV irradiation experiments, an EC Plan-Neofluar 100×/1.30 Oil Iris objective was used. The microscope is equipped with full environmental control, and imaging took place at 37 °C. EGFP-tagged HK13 and mScarlet-tagged short EPPK1 were illuminated with a 488 nm laser (10% intensity, exposure time 150 ms) and a 561 nm laser (25% intensity, exposure time 200 ms), respectively. The signal was detected with a sCMOS: 2× pco.edge 4.2 camera system (2048 × 2048 pixel, 6.45 µm pixel size, 16 bit, 100 fps, QE 70%). Images were acquired with VisiView 5.0.0.11 acquisition software (Visitron Systems, Puchheim, Germany).

The lineage of immortalized mouse myoblasts C2C12 (ATCC® CRL1772™) was cultured at 37 °C and 5% CO₂ in growth medium (GM), composed of DMEM (Dulbecco’s Modified Eagle’s Medium) with high glucose and L-glutamine (Gibco), supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin, streptomycin, and amphotericin B solution (Gibco). Myogenic differentiation was induced in differentiation medium (DM), composed of DMEM, supplemented with 2% horse serum (Gibco) and 1% penicillin, streptomycin, and amphotericin B. For growth or differentiation conditions, the medium was replaced every 2 days.