Figure 3. Down-regulation of DDX3 enhances cell capabilities of self-renewal, chemoresistance, EMT and migration.

(a) DDX3 knockdown promoted self-renewal capability. Sphere formation assays of shLuc, shDDX3 #2 and shDDX3 #3 cells were performed and images of formed sphere were captured as described in the Materials and methods. Scale bar represents 100 μm. The primary spheres of these cells were further dissociated into single cells by trypsinization and subjected to another round of sphere formation assay. The numbers of primary and secondary spheres in shDDX3 #2 and shDDX3 #3 cells were transformed into fold change relative to that of shLuc cells. (b) Cells with decreasing DDX3 expression were more viable upon conventional anti-cancer drugs treatment. shLuc, shDDX3 #2 and shDDX3 #3 cells were treated with different concentrations of doxorubicin (0, 0.125, 0.25 and 0.5 μg/ml; left panel) or 5-fluorouracil (0, 2, 4 and 8 μg/ml; right panel), and cell viability was determined by MTT assay. Formazan absorbance at 550 nm of untreated cells was arbitrarily set as 100% viable. Viability of shLuc, shDDX3 #2 and shDDX3 #3 cells at each drug concentration was relative to that of corresponding untreated cells. (c) DDX3 knockdown promoted EMT. Microscopy of shLuc, shDDX3 #2 and shDDX3 #3 cells are shown. Scale bar is equal to 100 μm. Cell lysates (50 μg) of shLuc, shDDX3 #2 and shDDX3 #3 cells were analyzed by western blotting with antibodies against DDX3, E-cadherin, fibronectin and β-actin. (d) Down-regulation of DDX3 promoted cell mobility. Migration assays of shLuc, shDDX3 #2 and shDDX3 #3 cells were carried out and images of Giemsa-stained migrated cells were captured. Scale bar represents 100 μm. The number of migrated cells in shDDX3 #2 and shDDX3 #3 cells were relative to that in shLuc cells. All experiments were repeated at least three times, and the error bar indicated ± 1 s.d. of the mean. Statistical analyses were carried out using t test (*p < 0.05; **p < 0.01; ***p < 0.001).