Apoptosis assay

Cellular apoptosis was measured using Caspase-Glo® 3/7 assay (Promega Corporation, Madison, WI, USA, Cat# G8091). Human cardiomyocytes were seeded as described above in an opaque white-walled 96-well plate. The next day, the cells were pre-treated with 500 nM ATRA, 1000 nM ATRA, or the vehicle (DMSO). After 24 hours, the cells were treated with the respective ATRA dose or vehicle (DMSO) and 0 or 10 μM doxorubicin concentration for another 24 hours. The 10 μM concentration of doxorubicin was selected based on the doxorubicin IC₅₀ (half-maximal inhibitory concentration) measured in this study and to maintain physiological relevance [29, 30]. The assay was performed according to the manufacturer’s instructions and was read on POLARstar Omega plate reader (BMG Labtech) by measuring luminescence.