Fig. 5

Role of EID3 in radiotherapy- or chemotherapy-triggered biogenesis of cancer stem cells. A and B Silencing of APLF, EID3, EME2, NPAS2 and POLN in cisplatin-induced, doxorubicin-induced or X-ray-induced GCSCs. GCNSCs were treated with cisplatin, doxorubicin or X-ray and transfected with gene-specific siRNA. As a control, siRNA control was included in the transfection. Thirty-six hours after transfection, the gene expression levels in GCSCs were evaluated using quantitative real-time PCR (**p < 0.01) (A) and Western blot (B). C and D Impact of gene silencing on the expression of stemness genes in cisplatin-induced, doxorubicin-induced or X-ray-induced GCSCs. The gene expression levels were examined 36 h after siRNA transfection with quantitative real-time PCR (**p < 0.01) (C) and Western blot (D). E Influence of gene knockdown on the tumorsphere formation capacity of cisplatin-induced, doxorubicin-induced and X-ray-induced GCSCs. GCSCs were transfected with gene-specific siRNA. As a control, siRNA control was included in the transfection. At the 7th day, the tumorsphere formation capability was examined. F Impact of EID3 silencing on the expressions of stemness genes in GCNSCs without DNA damage. EID3-siRNA was transfected into GCNSCs without DNA damage. Thirty-six hours later, the expression levels of stemness genes in GCNSCs were examined with Western blot. β-tubulin was used as a control. G Effects of EID depletion on the expressions of stemness genes in radiotherapy- or chemotherapy-treated GCNSCs. EID3 was knocked down in cisplatin-, doxorubicin- or X-ray-treated GCNSCs, followed by the examination of stemness genes’ expressions using Western blot. β-tubulin served as a control. H The protein bound to EID3. Coimmunoprecipitation (Co-IP) assay was conducted using EID3-specific antibody. The mouse IgG was used as a control. The Co-IP products were examined by SDS-PAGE with Coomassie staining. The proteins identified by mass spectrometry were indicated with arrows. M, protein marker. I Western blot analysis of the Co-IP products using EID3-specific antibody or the mouse IgG. β-tubulin was used as a control. J Effects of EID3 silencing and rescue on the NAMPT-mediated Wnt signaling pathway. The expression levels of the key components of Wnt pathway in EID3-silenced or rescued cisplatin-induced, doxorubicin-induced and X-ray-induced GCSCs were examined by quantitative real-time PCR. As a control, siRNA control was included in the experiments. The statistical significance of difference between treatments was indicated by asterisks (**p < 0.01). K Western blot analysis of the impact of EID3 silencing and rescue on the NAMPT-mediated Wnt signaling pathway. L Impact of EID3 silencing and/or NAMPT inhibition on the Wnt signaling pathway. In cisplatin-induced, doxorubicin-induced or X-ray-induced GCSCs, the expression of EID3 was knocked down and/or the NAMPT activity was inhibited by a specific inhibitor FK866. Then, the expression levels of the key components of Wnt pathway were evaluated by quantitative real-time PCR (**p < 0.01). M Western blot analysis of the impact of EID3 silencing and/or NAMPT inhibition on the Wnt pathway in cisplatin-induced, doxorubicin-induced or X-ray-induced GCSC. β-tubulin was used as a control. (N) Correlation analysis between EID3 and the Wnt pathways. The correlation analysis was carried out based on the cBioportal datasets of gastric cancers (https://www.cbioportal.org/). O Impact of EID3 silencing and rescue on the tumorsphere formation capacity of the radiotherapy- or chemotherapy-induced GCSCs. The cisplatin-induced, doxorubicin-induced or X-ray-induced GCSCs were transfected with EID3-siRNA and/or the plasmid expressing EID3 (EID3 rescue). At the 7th day after transfection, the percentage of tumorsphere formation of cells was evaluated (**p < 0.01). P Effects of EID3 silencing or rescue on the expressions of stemness genes in cisplatin-induced, doxorubicin-induced and X-ray-induced GCSCs. GCSCs were transfected with EID3-siRNA and/or the plasmid expressing EID3. Thirty-six hours after transfection, the expression levels of stemness genes in cells were examined using Western blot. β-tubulin was used as a control. Q Role of the Wnt signaling pathway in the EID3-mediated stemness of radiotherapy- or chemotherapy-induced GCSCs. The cisplatin-induced, doxorubicin-induced and X-ray-induced GCSCs were treated with FK866 (50 nM) or ICG001 (10 μM) for 24 h. Subsequently, the cells were subjected to Western blot analysis. β-tubulin served as a control. R Model for the radiotherapy- or chemotherapy-triggered biogenesis of cancer stem cells from cancer non-stem cells