Mt‐Keima‐based mitophagy assay

For the detection of mitophagy, HeLa cells stably expressing mt‐Keima transiently transfected with the plasmid as indicated, and MITOL WT and KO HeLa cells stably expressing HA‐Parkin and mt‐Keima were treated with 10 μM CCCP for 8 h. For measurement of mKeima, a BD LSR II Flow Cytometer was used to detect the dual‐excitation ratiometric pH at 488 (pH 7) and 561 (pH 4) nm lasers with 695/40 and 575/26 nm emission filters. For each sample, 30,000 cells were counted and analyzed using FlowJo software (version 10.7.1).