Figure 2: Simvastatin combined with tamoxifen upregulates γH2AX and induces DNA damage in tamoxifen-resistant breast cancer cells.

(A) MCF7 and T47D cells were cultured at different doses (0 μM, 10 μM and 20 μM) of simvastatin. After treatment for 24 h, the cells were lysed, and a western blot was performed. (B) Four groups of MCF7 TamR and T47D TamR cells were cultured with a control solvent, 4-OH-tamoxifen (5 μM), simvastatin (20 μM), or 4-OH-tamoxifen (5 μM) plus simvastatin (20 μM). After treatment for 24 h, the cells were lysed, and a western blot was performed. (C) Four groups of MCF7 TamR and T47D TamR cells were cultured as described above. After treatment for 24 h, the cells were lysed, and a western blot was performed. (D) Four groups of MCF7 TamR and T47D TamR cells were cultured as described above. After treatment for 24 h, the cells were fixed, and immunofluorescence was performed. The images were obtained by fluorescence confocal microscopy; the bars represent 10 μm. (E) Four groups of MCF7 TamR cells were cultured as described above. After treatment for 24 h, comet assays were performed under alkaline conditions. Images were obtained by fluorescence confocal microscopy; the bar represents 100 μm. (H) Quantitative analysis of the DNA content in the comet tail relative to the total DNA content in each group of cells. Fifty cells were scored per group. *p < 0.05. (F) Four groups of MCF7 TamR and T47D TamR cells were cultured as described above. After treatment for 24 h, chromosome fragility analyses were performed, and representative images are shown. The chromosome gaps and breaks (indicated as arrows) were scored. Two metaphases were analyzed for each cell group. (G) Four groups of MCF7 TamR and T47D TamR cells were cultured as described above. After treatment for 24 h, the cells were lysed, and a western blot was performed.