Novel therapeutic diiminoquinone exhibits anticancer effects on human colorectal cancer cells in two-dimensional and three-dimensional in vitro models

Figure 6 Diiminoquinone induced apoptosis and inhibited proliferation in colon cancer stem/progenitor cells. Representative immunofluorescence imaging of control and diiminoquinone (DIQ)-treated HCT116 and HT29 spheres collected at generation 1. A-D: Spheres stained for cytokeratin (CK)8 (green) and CK19 (red) (A), KI67 (B), CD44 (C), and gamma H2A histone family member X (γH2AX) (D) were obtained using confocal microscopy. The nuclei were stained with anti-fade reagent Fluorogel II with 4’,6-diamidino-2-phenylindole (DAPI). The quantification of the intensity of CK8, CK19, CD44, and Ki67 stain in control and DIQ-treated spheres was performed using Carl Zeiss Zen 2012 image software. Stain intensity was normalized to size. Scale bar 20 μm; E: Analysis of p53, p21, CD133, β-catenin, proliferating cell nuclear antigen (PCNA), p-ERK, ERK, p-AKT, and AKT protein expression in HCT116 and HT29 generation 1 spheres upon treatment is shown. GAPDH served as an internal control. Bands were detected by enhanced chemiluminescence using ChemiDoc MP Imaging System. Fold expression changes normalized to GAPDH and to total ERK and total AKT in the cases of p-ERK and p-AKT expression, respectively, are given. Data represent an average of three independent experiments and is reported as mean ± standard error of the mean (^aP < 0.05, ^bP < 0.01, ^cP < 0.001).