Fig. 2

Radiotherapy and chemotherapy promote DNA repair and biogenesis of cancer stem cells. A Expressions of DNA repair genes in radiotherapy- or chemotherapy-treated gastric cancer non-stem cells (MKN-45, GCNSCs). The cells without any treatment were used as controls. The gene expression levels were examined with quantitative real-time PCR. B Western blot analysis of gene expression. β-tubulin was used as a control. C Expression profiles of stemness genes in radiotherapy- or chemotherapy-treated GCNSCs. The expressions of stemness genes in cisplatin-, doxorubicin- or X-ray-treated GCNSCs were determined by quantitative real-time PCR. The cells without any treatment were used as controls. D Upregulation of stemness genes in radiotherapy- or chemotherapy-treated GCNSCs. The cisplatin-, doxorubicin- or X-ray-treated GCNSCs were subjected to Western blot. β-tubulin was used as a control. E Percentage of tumorsphere formation of radiotherapy- or chemotherapy-treated GCNSCs. GCNSCs were treated with cisplatin, doxorubicin or X-ray. After culture for 7 days, the tumorsphere formation efficiency of radiotherapy- or chemotherapy-treated GCNSCs was evaluated. The data were the average values of three independent experiments. F Tumorsphere formation of a single tumor cell. A single cell from a tumorsphere of radiotherapy- or chemotherapy-treated GCNSCs was subjected to tumorsphere formation assay. At different time after culture, the tumorsphere was examined. Scale bar, 20 μm. In all panels, the significant differences between treatments were indicated with asterisks (*p < 0.05; **p < 0.01). G Immunofluorescence analysis of the radiotherapy- or chemotherapy-treated GCNSCs. GCNSCs (sorted from MKN-45) were treated with cisplatin, doxorubicin or X-ray for 48 h. As a control, GCNSCs alone were included in this assay. Subsequently, the cells were subjected to immunofluorescence staining to examine the stemness markers (Lgr5 and Klf4). Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). Representative images of immunofluorescence staining were presented. Scale bar, 5 μm