Fig. 5
Requirement of YB-1 for the reversion of differentiated cancer cells into cancer stem cells. a Expressions of stemness-related transcription factors in YB-1 wild-type, YB-1 knockout, and YB-1 rescue cancer stem cells. Quantitative real-time PCR (left) and Western blot (right) were conducted to detect the expression levels of the 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). b Overexpressions of stemness-related transcription factors in YB-1 knockout cancer stem cells. The constructs of the other 4 (SOX2, POU3F2, OCT-4, and OLIG1) and 5 (SOX2, SALL2, OCT-4, POU3F2, and Bmi-1) transcription factors were transfected into the YB-1 knockout melanoma stem cells and breast cancer stem cells, respectively. At 36 h after transfection, quantitative real-time PCR (left) and Western blot (right) were performed to examine the expression levels of genes. The experiments were biologically repeated for three times (**, p < 0.01). c Influence of stemness-related transcription factors on the tumorsphere formation capacity of cancer stem cells. A single cell was plated into a 96-well plate. The cells were cultured for 10 days and examined under a light microscope at days 1, 3, 5, and 10. Scale bar, 50 μm. The experiments were repeated for three times. d Effects of simultaneous expressions of stemness-associated transcription factors on the expressions of stemness genes in cancer stem cells. At 36 h after transfection, quantitative real-time PCR (above) and Western blot (below) were conducted to evaluate the expression levels of stemness genes (Nanog, ALDH1, and ABCG2). The experiments were performed in triplicate (**, p < 0.01). e Impact of YB-1 rescue and simultaneous overexpressions of the other four or five transcription factors on the expression profiles of differentiation genes in YB-1 knockout melanoma stem cells and breast cancer stem cells. At 48 h after transfection, quantitative real-time PCR (left) and Western blot (right) were used to evaluate the expression levels of differentiation genes. The experiments were repeated for three times (**, p < 0.01)