Figure 7.

HI-511, a dual-target inhibitor for both AURKB and BRAF V600E, affects cell growth, apoptosis, and protein expression levels in A375 and A375R melanoma cells. (A) Melanocytes (NHEM; 1 × 10⁴/well) were seeded into 96-well plates. After incubation overnight, cells were treated with the indicated concentrations of HI-511 and incubated for 24 h or 48 h. Viability was estimated using the MTS assay as described in Materials and Methods. (B, C) HI-511 inhibited anchorage-independent cell growth. A375 and A375R cells (8 × 10³/well) were seeded into 6-well plates with 0.3% Basal Medium Eagle agar containing 10% FBS and different concentrations of HI-511 and then cultured for 2 weeks. Colonies were scored under a microscope using the Image-Pro PLUS (v6.) software program. (D) Representative colonies images of A375 and A375R. (E, F) Effects of HI-511 on the activation of histone H3, MEK, ERKs, and AKT were detected by Western blot in A375 and A375R cells. (G, H) A375 and A375R cells (2.5 × 10⁵/well) were incubated with HI-511 (1.25, 2.5, or 5 µM) or vehicle control for 48 h. Cells were collected and apoptosis was detected using flow cytometry and Annexin V, propidium iodide staining. (I, J) The cells were incubated with HI-511 or vehicle control for 48 h, then the effect of HI-511 on apoptosis-associated protein expression was determined by Western blot. Statistical significance was determined by one-way ANOVA. The asterisks indicate a significant change compared with untreated control cells (p < 0.01; ***, p < 0.001).