Journal: Cell Death & Disease

Article Title: Discovery of a new molecule inducing melanoma cell death: dual AMPK/MELK targeting for novel melanoma therapies

doi: 10.1038/s41419-020-03344-6

Figure Lengend Snippet: 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.

Article Snippet: Adenoviruses encoding a dominant-negative form (Ad AMPK-DN) of subunits α1 and α2 of AMPK were a generous gift of Dr. Foufelle (INSERM, UMR-S 872, Paris, France).

Techniques: Control, Membrane, Flow Cytometry, Western Blot, Cell Fractionation, Transduction, Dominant Negative Mutation

Journal: Cell Death & Disease

Article Title: Discovery of a new molecule inducing melanoma cell death: dual AMPK/MELK targeting for novel melanoma therapies

doi: 10.1038/s41419-020-03344-6

Figure Lengend Snippet: A Scatter plot representing the percentage of kinase inhibition and their Z′ factor measured during two independent in vitro kinome assay with 1 μM CRO15. B Immunofluorescence pictures of CT26 cancer cells treated with 5 μM CRO15 for 2 h. MELK was labeled with a specific antibody (in red), CRO15 was coupled to a fluorescent tag (in green) and nuclei were stained with DAPI (in blue). C In vitro test of kinase activity using recombinant MELK protein treated with CRO15 with the MELK inhibitor OTS167. D A375 cells were infected with MELK adenovirus for 24 h. For the pull down assay, cells lysates were incubated overnight with 100 μM Biotin-CRO15 or its inactive analog MTF465. For Biotin precipitation, cells were treated for 2 h with 20 μM Biotin-CRO15 or MTF465 before lysis and western blot analysis with the indicated antibodies. One representative experiment of three is shown. E and F A375 cells were treated with 5 μM CRO15 with the indicated durations, and lysates were analyzed by western blotting using the indicated antibodies. One representative experiment of three is shown. G A375 cells were transfected with siRNA directed against AMPK α1 and AMPK α2 (20 nM each) or 40 nM of control siRNA. Twenty-four hours later, cells were infected with a MELK adenovirus or a control adenovirus before treatment with 5 µM of CRO15. After 24H, viable cells were counted using the trypan blue dye exclusion method ( n = 3) (right panel). In parallel, cell lysates were analyzed by western blotting with the indicated antibodies (left panel). One representative experiment of three is shown.

Article Snippet: Adenoviruses encoding a dominant-negative form (Ad AMPK-DN) of subunits α1 and α2 of AMPK were a generous gift of Dr. Foufelle (INSERM, UMR-S 872, Paris, France).

Techniques: Inhibition, In Vitro, Immunofluorescence, Labeling, Staining, Activity Assay, Recombinant, Infection, Pull Down Assay, Incubation, Lysis, Western Blot, Transfection, Control

Journal: Cell Death & Disease

Article Title: Discovery of a new molecule inducing melanoma cell death: dual AMPK/MELK targeting for novel melanoma therapies

doi: 10.1038/s41419-020-03344-6

Figure Lengend Snippet: A A375 cells were treated with 5 μM CRO15 for the indicated duration. Lysates were analyzed by western blotting with the indicated antibodies. One representative experiment of three is shown. B Immunofluorescence pictures of A375 melanoma cells treated with DMSO, 5 μM CRO15 or 400 nM of the autophagy inducer rapamycin for 6 h. LC3B was labeled with antibody (green), and DNA was visualized with DAPI (blue). C A375 melanoma cells were treated for 24 h with 5 μM CRO15, 5 μM PLX4032 or with 1 μM of the pan-kinase inhibitor staurosporine for 6 h used as positive control of apoptosis induction. Cells were analyzed by flow cytometry using a specific antibody against Caspase 3 active protein conjugated with FITC (upper panel) or Annexin V conjugated with ALEXA and DAPI (lower panel) or against. * p < 0.05; ** p < 0.01; *** p < 0.001. D A375 cells were infected with dominant negative adenoviruses against α1 and α2 AMPK subunits for 24 h before transfection with sip53 for another 24 h. Cells was determined by trypan blue staining after a 24 h treatment with 5 μM CRO15 or DMSO control. E A375 cells were infected with dominant negative adenoviruses against α1 and α2 AMPK subunits for 24 h and were transfected with sip53 for another 24 h. Cells were treated with 5 μM CRO15 for the indicated durations, and lysates were analyzed by western blot with the indicated antibodies. One experiment of three is shown. F A375 cells were treated with 5 μM CRO15 for the indicated durations, and lysates were analyzed by western blotting with the indicated antibodies. One representative experiment of three is shown. G A375 cells were transfected with siREDD1 for 24 h and then treated with 5 μM CRO15 for 24 h before cell viability was determined using the trypan blue exclusion method. H . A375 cells were transfected with siREDD1 for 24 h and then treated with 5 μM CRO15 for the indicated duration. Lysates were analyzed by western blotting with the indicated antibodies. One representative experiment of three is shown. For D and G , the results are expressed as percentages of the control and data given as the means ± SEM of three independent experiments performed in triplicate. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Adenoviruses encoding a dominant-negative form (Ad AMPK-DN) of subunits α1 and α2 of AMPK were a generous gift of Dr. Foufelle (INSERM, UMR-S 872, Paris, France).

Techniques: Western Blot, Immunofluorescence, Labeling, Positive Control, Flow Cytometry, Infection, Dominant Negative Mutation, Transfection, Staining, Control