Fig. 5. CK potentiates mtROS and loss of mitochondrial membrane potential but not cellular ROS generation.

a J774 cells were pre-incubated with 5 µM DCFDA prior to the respective treatments and DCFDA fluorescence was measured every 15 min for 1 h. b The relative amount of DCFDA fluorescence as calculated by percentage change from the baseline fluorescence after 1 h, following the treatments in a. c J774 cells were pre-incubated with 5 µM mitoSOX prior to the respective treatments and mitoSOX fluorescence was measured every 15 min for 105 min. d The relative amount of mitoSOX fluorescence as calculated by percentage change from the baseline fluorescence after 105 min, following the treatments in c. Experiments are representative of three independent experiments (n = 3). Error bars represent SD. Asterisks represent a significant difference (p < 0.05). e J774 cells were stimulated for 4 h with 500 µM ATP alone, or in combination with CK or CK and AZ10606120 (both 10 µM), with 3 mM ATP ± AZ10606120, or with CK alone. Staurosporine (5 µM) was used as a positive control. Cells were incubated with TMRM (100 nM) for the final 30 min of the treatment time (4 h or 24 h) and fluorescent images were taken. f The percentage of cells positive for TMRM was identified by quantifying the number of cells positive for TMRM fluorescence in Fiji. Experiments are representative of three independent experiments (n = 3). Error bars represent SD. Asterisks represent a significant difference (p < 0.05). Scale bars are 50 µm.