MitoTracker Green FM staining and quantification

The mitochondrial density was measured using MitoTracker probes (Cell Signaling Technologies; #9074P). This probe is cell permeable and contains a mildly thiol-reactive chloromethyl moiety for mitochondrial labeling. Phoenix hiPSC-CMs were seeded on 48-well culture plates at a density of 100,000 cells per well. The hiPSC-CMs were transduced with AAV6 virus expressing hTERT and, as control, the empty virus in presence and absence of 1 μM doxorubicin stress in normal maintenance medium for 48 h. MitoTracker Green FM (500 nM) and Hoechst 33342 (1:1,000, Thermo Fisher; #62249) working solution was prepared in Opti-MEM reduced serum media (Gibco; #51985-026) and loaded on the live cells for 15 min at 37°C. The cells were washed once with Opti-MEM reduced serum media. MitoTracker Green FM fluorescence intensity was measured at emission of 530 nm (excitation 485 nm). Each group consisted of 5 wells, and the fluorescence intensity of the entire well with 100,000 cells was measured. The fluorescence intensity and images were acquired using Cytation 1 (Biotek).