Mitoros

Mitochondrial functions were examined using fluorescent probes. Cells were incubated with the probes for 30 min at 37 °C and then imaged using a BZ-X710 All-in-One fluorescence microscope (KEYENCE, Osaka, Japan). We used MitoROS (mtROS) (10 μM, AAT Bioquest Inc., Sunnyvale, CA, USA) and dihydrorhodamine 123 (DHR) (10 μM, Sigma-Aldrich) to assess oxidative stress, mitoGreen (mtGR) (100 nM, PromoCell GmbH, Heidelberg, Germany) to assess mitochondrial volume, tetrathylrhodamine ethyl ester (TMRE) (200 nM, Sigma-Aldrich) to assess mitochondrial membrane potential, and mitoFerrogreen (mtFG) (20 nM, Dojindo, Kumamoto, Japan) to assess mitochondrial iron (Fe²⁺).

BJ cells were treated with vehicle or abemaciclib (1 μM ‐ 4 times in 24 h) and harvested for the MitoROS (AAT Bioquest, 16052) staining. The cells were stained for 10 min at 37°C in darkness. Following the staining, cells were centrifuged at 400 g for 5 min and cell pellet was resuspended in serum‐free DMEM for analysis by flow cytometry.

Mitochondrial functions were examined using fluorescent probes. After treatment with or without BBR (25 μM), cells were incubated with the probes for 30 min at 37 °C and then photographed using an All-in-One fluorescence microscope (KEYENCE). We used MitoROS (mitochondrial superoxide) (10 μM, AAT Bioquest Inc., Sunnyvale, CA, USA) to assess oxidative stress, mitoGreen (100 nM, PromoCell GmbH, Heidelberg, Germany) to assess mitochondrial volume, tetramethylrhodamine ethyl ester (TMRE) (200 nM, Sigma-Aldrich) to assess MMP, and mitoFerrogreen (20 nM, Dojindo, Kumamoto, Japan) to assess mitochondrial iron (Fe²⁺). Mitophagy was detected using a Mitophagy Detection Kit (Dojindo), according to the manufacturer’s instructions.