Lysoview 650

Live mCherry-EGFP-SYNJ2B-TM H9c2 cells grown in MatTek dishes were stained with LysoView 650 (Biotium, 70059) for 30 min and imaged using the Airyscan FAST mode of the LSM880, utilizing line-wise switching between tracks to avoid time delay between channels. Cells were maintained in a humidified stage-top incubator at 37°C and 5% CO₂ during imaging. Timelapse series (500 frames in total) were recorded using a 40× NA1.2 water immersion objective lens and a zoom factor of 4.0, with optimized scan settings for subsequent Airyscan processing (50 nm pixel size). The pixel dwell time was 0.73 μs, resulting in an individual frame time of 1.01 s. Laser excitation at 488 nm, 561 nm and 633 nm was used for EGFP, mCherry, and LysoView 650, respectively. The resulting raw files were processed using automatic settings (strength 6.0) in ZEN ver. 2.3 (Carl Zeiss Microscopy).

Rat cardiomyoblast H9c2 cells (Sigma-Aldrich, 88092904) were cultured in high-glucose (4.5 g/L) Dulbecco’s Modified Eagle Medium (DMEM; Sigma-Aldrich, D5796) with 10% Fetal Bovine Serum (FBS) and 1% streptomycin/penicillin (Sigma-Aldrich, P4333). For glucose deprivation and adaptation to galactose, the cells were grown in DMEM without glucose (Gibco, 11966–025) supplemented with 2 mM L-glutamine (Sigma-Aldrich, G7513) 1 mM sodium pyruvate (Sigma-Aldrich, S8636), 10 mM galactose (Sigma-Aldrich, G5388), 10% fetal bovine serum (Sigma-Aldrich, F7524) and 1% streptomycin-penicillin (Sigma-Aldrich, P4333). The cells were adapted to galactose for at least 7 days before the experiments. Stable H9c2 cells (see below) were grown in the same medium with the addition of 1 μg/ml of puromycin (InvivoGen, ant-pr-1). For hypoxic conditions, the cells were incubated at 0.3% O₂ for 2, 4, 6 or 16 h. For labeling of lysosomes, the cells were treated with 50 or 100 nM LysoTracker Deep Red (ThermoFisher Scientific, L12492) for 30–40 min or with LysoView 650 (Biotium, 70059). Cells were treated as indicated with 0.2 μM bafilomycin A₁ (BafA1 from Streptomyces griseus; Sigma-Aldrich, B1793) or 10 μg/ml pepstatin A (Sigma-Aldrich, P5318) and 10 μg/ml E64d (Sigma-Aldrich, E8640). All cell lines were maintained at 37°C and under 5% CO₂.