Fig. 2
Requirement of YB-1 for the stemness of cancer stem cells. a The expression of YB-1 in YB-1 knockout cancer stem cells. The YB-1 knockout melanoma stem cells (MDA-MB-435) and breast cancer stem cells (MCF-7) were transfected with pcDNA-YB-1 plasmid containing YB-1 coding sequence to express YB-1. At different time after transfection, the YB-1 protein was detected with Western blot. β-tubulin was used as a control. The images were the representatives of three experiments. b The effect of YB-1 on cell viability of melanoma stem cells and breast cancer stem cells. YB-1 knockout, YB-1 wild-type, and YB-1-rescue cancer stem cells were seeded into a 96-well plate at 1 × 103 cells/well and cultured for various time, followed by the examination of cell viability. The experiments were biologically repeated for three times. Student’s t test was used to assess the statistical significance of difference between treatments (*, p < 0.05, **, p < 0.01). c The examination of cell number. YB-1 knockout, YB-1 wild-type, and YB-1-rescue cancer stem cells were seeded into a 6-well plate at 1 × 104 cells/well. At different time after treatments, the numbers of melanoma stem cells (MDA-MB-435) and breast cancer stem cells (MCF-7) were evaluated. The experiments were carried out in triplicate. The statistical significance of difference between treatments were evaluated using Student’s t test (*, p < 0.05, **, p < 0.01). The time point 0 h represented the number of inoculated cells. d The influence of YB-1 on cell cycle of cancer stem cells. YB-1 knockout, YB-1 wild-type, and YB-1-rescue cancer stem cells were cultured for 48 h and then the percentage of cells (1 × 104) in the G1 phase was examined with flow cytometry. The experiments were performed in three times. Student’s t test was used to assess the statistical significance (*, p < 0.05, **, p < 0.01). e The impact of YB-1 knockout on caspase 3/7 activity of cancer stem cells. Melanoma stem cells (MDA-MB-435) and breast cancer stem cells (MCF-7) were seeded into a 96-well plate at 1 × 104 cells/well subjected to the detection of caspase 3/7 activity, following culture for 48 h. The experiments were conducted for three times. Student’s t test was used to evaluate the statistical significance of difference between treatments (*, p < 0.05; **, p < 0.01). f Examination of apoptosis using Annexin V assays. Melanoma stem cells (MDA-MB-435) and breast cancer stem cells (MCF-7) were seeded into a 6-well plate at 1 × 105 cells/well and culture for 48 h. Then, apoptosis of cancer stem cells were examined by flow cytometry. The experiments were biologically repeated for three times. The statistical significance of difference between treatments was assessed by Student’s t test (**, p < 0.01). g Influence of YB-1 knockout on the tumorsphere formation capacity of melanoma and breast cancer stem cells. At different times (1, 3, 5, and 10 days) after culture, the cells were examined under a light microscope. The statistical analysis of the diameter of tumorspheres was indicated on the right (**, p < 0.01). Scale bar, 50 μm. h Effects of YB-1 knockout on the expressions of stemness genes in MDA-MB-435 melanoma stem cells and MCF-7 breast cancer stem cells. At 48 h after cancer stem cell culture, quantitative real-time PCR was used to evaluate the expression levels of stemness genes. The experiments were performed in three times. Student’s t test was used to assess the statistical significance (**, p < 0.01). i Impact of YB-1 knockout on the expressions of differentiation genes in cancer stem cells. At 48 h after culture, the expression levels of differentiation genes in cancer stem cells were examined with quantitative real-time PCR. The experiments were repeated for three times. Student’s t test was used to assess the statistical significance of difference (**, p < 0.01)