Fig. 2: CRO15 induces a mitochondrial disorder and activates AMPK pathway.

A Measurement of mitochondrial function in A375 melanoma cells with a Seahorse XF Cell Mito stress test after 6 h of treatment with 5 μM CRO15, 10 mM metformin or DMSO used as control. At 20 min, cells are treated with oligomycin, an inhibitor of complex V of respiratory chain; at 50 min, with FCCP, which disrupts the mitochondrial membrane potential; and at 80 min, with Rotenone and antimycin A, inhibitors of complex I and III, respectively. B A375 cells were treated with 5 μM CRO15 (6H), 10 mM metformin (6H) for or 20 μM CCCP (20 min). Measurement of mitochondrial potential membrane was performed by flow cytometry with a TMRE probe. C Lysates from A375 melanoma cells exposed for the indicated durations to CRO15 were analyzed by western blotting using the indicated antibodies after cell fractionation. One representative experiment of three is shown. D Lysates from A375 cells treated with 5 μM CRO15 for the indicated durations were analyzed by western blotting using the indicated antibodies. One representative experiment of three is shown. E Lysates form A375 cells transduced with dominant negative AMPK subunits α1 and α2 for 24 h and treated with 5 μM CRO15 were analyzed by western blotting using the indicated antibodies. One representative experiment of three is shown. F Quantification of cell viability from E using trypan blue exclusion method. Results are expressed as percentages of DMSO control and data given as the means ± SEM of three independent experiments performed in triplicate. *p < 0.05; **p < 0.01; ***p < 0.001.