Figure 5.

Combination effects of RM + DOX (1:50) on MCF-7 cell cycle and apoptosis. (A) DNA content analysis of MCF-7 treated singly or concurrently with RM and DOX for 72 h. The histograms are representative of two independent trials. The percentages reported are mean ± SEM and are represented also in bar graphs (right). High concentration of the combination (25 nM RM + 1250 nM DOX) induced an S and G2/M arrest, while lower concentrations of the combination (6.25 nM RM + 313 nM DOX and 1.56 nM RM + 78 nM DOX) only induced a G2/M arrest. Compared to the single drug treatments, a slight decrease in G2/M and a consequent increase in sub-G1 (dead cells) were observed after the combination treatment. MCF-7 treated with the vehicles 0.1% DMSO and sdH₂O served as negative controls (neg). * p < 0.05, ** p < 0.001, *** p < 0.0001, and + p < 0.05, ++ p < 0.001, +++ p < 0.0001 vs. RM alone and DOX alone, respectively (one-way ANOVA/Bonferroni). (B) Annexin V/PI flow cytometry of MCF-7 treated singly or concurrently with RM and DOX for 30 h. The dot plots are representative figures of two independent trials. (C) Hoechst 33342 staining of nuclei after 24 h of exposure to the compounds. Shown are representative images of two independent trials. Minimum of 200 cells were counted manually and the number of apoptotic cells is reported in bar graphs (bottom). White arrows indicate brightly stained condensed nuclei. Bars are mean + SEM of three independent trials performed in quadruplicates. * p < 0.05, ** p < 0.001, and *** p < 0.0001. Scale bar = 20 μm.