Compounds A and I induce apoptosis selectively and Compound A interacts positively with tamoxifen. (A) A375 melanoma cells and (B) noncancerous normal human fibroblasts (NHF) were treated with the indicated compounds for 48 h then stained with Annexin V and PI to quantify apoptosis, as described in the materials and methods section. Image based cytometry was utilized to assess apoptosis. The y-axis represents the percent of cells positive for Annexin V (green), PI (red), Annexin V and PI (orange), or cells negative for Annexin V and PI (blue). Values are expressed as mean ± SD from three independent experiments. (C) A375 melanoma cells and (D) NHF cells were treated with Compound A and curcumin alone and in combination with tamoxifen for 48 h. Please note, the data of Compound A and curcumin alone shown in Figure 4A,B were used again in Figure 4C,D, respectively along with the combination treatments for direct comparison. *p < 0.05 vs. DMSO control (comparison of viable cells only); **p < 0.01 vs. DMSO control (comparison of viable cells only); ***p < 0.001 vs. DMSO control (comparison of viable cells only); ****p < 0.0001 vs. DMSO control (comparison of viable cells only); #p < 0.05 vs. 0.1 μM Compound A alone (comparison of viable cells only); $p < 0.05 vs. 0.25 μM Compound A alone (comparison of viable cells only); ^p < 0.05 vs. 5 μM CUR alone (comparison of viable cells only); @p < 0.05 vs. tamoxifen treatment alone (comparison of viable cells only). (E) A375 micrographs at 48 h. Top: Bright field images at 400× magnification. Bottom: Fluorescent images stained with PI (red) and Hoechst (blue). Scale bar = 100 μm. Micrographs are representative of three independent experiments. A = Compound A, I = Compound I, CUR = Curcumin, TAM = Tamoxifen.