Lab tek 2 chamber glass slides

To measure cytosolic ROS in live iPSC-CMs, cells were loaded with 5 µM CellROX™ Deep Red Reagent (Molecular Probes, OR, USA) and 2 µM Hoechst 33,342 (Invitrogen) at 37 °C for 30 min, and then washed 3 times with PBS. For mitochondrial morphology and ROS detection, iPSC-CMs were loaded with MitoTracker^® Green FM (Molecular Probes) at 200 nM and MitoSOX™ Red (Molecular Probes) at 5 µM for 15 min at 37 °C, then washed gently 3 times with warm buffer before imaging. For the treatment of maturation medium (MM), iPSC-CMs were switched to MM for 48 h before functional analysis. For the treatment of compounds, N-Acetylcysteine amide (NACA) was used at 5 mM and rapamycin used at 0.5 µM final concentration, respectively. For live cell imaging analysis, all the cells were seeded on Lab-Tek^® II chamber glass slides (Nunc) or glass-bottom 35 mm dishes with 14 mm microwell and No. 1.5 cover glass (MatTek, Ashland, MA, USA).

Cell proliferation was measured using the BrdU Cell Proliferation Kit (Millipore, Billerica, MA, USA) according to the manufacturer’s instructions. Human CECs were seeded at a density of 7,500 cells/well in FNC-coated 8-well Lab-Tek II chamber glass slides (Nunc, Naperville, IL, USA). To establish the scraping wound model, cells were scratched with a 200 μL pipette in the middle of wells 24 hours after seeding. For inhibitor treatments, human CECs were pre-incubated with either LY294002 (20 μM) or neomycin (1 mM) for 1 hour before and during exposure to RNase 5 (5 μg/mL for 24 hours). Subsequent steps are described in the Supplementary Methods.