Fig. 7: CRO15 induced death in melanoma cells resistant to BRAF inhibitors.
A Indicated melanoma cell lines were treated with 5 μM CRO15 or 5 μM PLX4032. After 48 h, cell viability was determined by Trypan blue exclusion method. The results are expressed as percentages of the corresponding control and data given as the means ± SEM of three independent experiments performed in triplicate. *p < 0.05; **p < 0.01; ***p < 0.001. B A375-resistant cells were treated with 5 μM CRO15 for the indicated durations. Lysates were analyzed by western blotting with the indicated antibodies. One representative experiment of three is shown. C Immunofluorescence images of A375-resistant melanoma cells treated with 5 μM CRO15 or 400 nM rapamycin for 6 h or DMSO control. LC3B was labeled with antibody (green), and the nuclei were visualized with DAPI (blue). D A375-resistant melanoma cells were treated for 24 h with 5 μM of CRO15 or PLX4032 for 24 h or with 1 μM staurosporine for 6 h. Cells were analyzed in flow cytometry with a specific antibody directed Annexin V (conjugated with ALEXA) or Caspase 3 active protein (conjugated with FITC). *p < 0.05; **p < 0.01; ***p < 0.001. E Female immune-deficient BALB/c nu/nu (nude) mice were inoculated subcutaneously with 1.0 × 106 A375-resistant cells. After 7 days, mice (n = 6 in each group) were treated with CRO15 (0.7 mg/mouse/day), PLX4032 (0.7 mg/mouse/day) or vehicle (Labrafil). Tumor growth curves were determined by measuring the tumor volume. The bars indicate the mean ± SEM. *p < 0.05; **p < 0.01. F Tumor weight of A375-resistant xenograft after mouse euthanasia. The bars indicate the mean ± SEM *p < 0.05; **p < 0.01.
