Fig. 3

Mechanism underlying the requirement of YB-1 for the stemness of cancer stem cells. a Functional classification of the downstream target genes of YB-1. b Motifs of DNAs binding to YB-1 protein. The promoter sequences bound with the YB-1 protein could be classified into 5 motifs. c Direct interaction between YB-1 protein and five motifs. After incubation of a motif with the YB-1 protein, the mixture was separated by 1% agarose gel and stained with ethidium bromide to visualize the DNA. The wedges indicated the concentration gradient of YB-1 protein used. The experiments were performed in triplicate. d Expressions of FZD1, p21, GLP-1, GINS1, and Notch2 genes in YB-1 knockout and rescue cancer stem cells. Quantitative real-time PCR was conducted to detect the expression levels of these genes. The experiments were repeated for three times. Student’s t test was used to assess the statistical significance of difference between treatments (**, p < 0.01). e Direct interaction between YB-1 and the promoters of YB-1 target genes. The plasmid containing a YB-1 target gene’s promoter and the control pRL-TK plasmid were co-transfected into YB-1 wild-type, knockout, or rescue cancer stem cells. At 36 h after transfection, the firefly luciferase activity and renilla luciferase activity were analyzed to evaluate the promoter activity. The experiments were conducted in triplicate. Student’s t test was used to evaluate the statistical significance (**, p < 0.01). f Proposed model of YB-1 regulating cancer stem cell proliferation and stemness