We have measured this change in mitochondrial membrane potential after treatment of cells with different doses of ATO and by labeling with very sensitive cationic carbocynine dye, JC-1. In control sample, healthy mitochondria showed high mitochondrial membrane potential (ψm) with intact membrane and accumulated in their matrix more JC-1 to form J- aggregates, showing intense fluorescence at 590 nm. Whereas in ATO treated cells, mitochondria showed lower ψm and less accumulation of JC-1 in their matrix leading to less formation of J-aggregates, and weak fluorescence at 590 nm (Figure 3A). We have also done confocal microscopy imaging of control and ATO-treated cells followed

by staining with JC-1 and DAPI. JC-1 monomer (530 nm) expression was activated by ATO treatment in #selleckchem randurls[1|1|,|CHEM1|]# a dose-dependent manner [Figure 3B (i-v)]. Figure 3 ATO changes mitochondrial membrane potential (Δψm). (A) ATO treatment was changed the mitochondrial membrane potential in a dose- dependent manner. [(B)(i-v)] There are three subsets of each treatment-DAPI (blue), JC-1 monomer (excitation 530 nm, green) and merged (blue/green). ATO treatment dose–dependently changed mitochondrial membrane potential and opened transition pores. It helped to release J-aggregate and continuously increased JC-1 monomer (green color) in a dose dependent manner in HL-60 cells.

Arsenic trioxide stimulates translocation of Bax and Cytochrome C Previous research has reported that Elafibranor oxidative stress activates translocation of pro-apoptotic proteins from cytosol to mitochondria and release of cytochrome C from mitochondria to cytoplasm inside cell [33]. We have checked ATO-induced translocation of pro-apoptotic protein, Bax from cytosol to mitochondria and cytochrome C from mitochondria to cytosol by labeling cells with Hoechst staining, mitochondria with mitotracker red and Bax as well as cytochrome C protein with green fluorescent antibody. Our results show that the amount of translocated Bax

inside mitochondria Atorvastatin [Figure 4 (i-v)] and cytochrome C protein in cytosol of ATO treated HL-60 cells increased in a dose-dependent manner [Figure 5A (i-v)]. We used green fluorescent tag anti-Bax and anti-cytochrome C antibody to recognize translocation of Bax and cytochrome C by immunocytochemistry and confocal imaging of cells. Figure 4 (i-v) Arsenic trioxide stimulates translocation of Bax protein. Each image set contains four subsets, a – cells stained with DAPI (blue); b – mitochondria stained with mitotracker red CMXRos (red, 250 nM); c – Bax protein tagged with fluorescent secondary antibody (green); and d – merged image of all previous three (a, b and c). Both immunocytochemistry and confocal imaging show translocation of pro-apoptotic protein, Bax from cytosol to mitochondria in a dose – dependent manner. Figure 5 Arsenic trioxide induces release of cytochrome C protein from mitochondria and activation of caspase 3.