Figure 4

(a) CD133-depleted partial CRISPR-Cas9 BAK-R T3 cells show decreased CD133 expression and are less invasive and metastatic than BAK-R-SC controls. CD133 positivity was determined by immunofluorescence staining in BAK-P, BAK-R, partial CRISPR-Cas9 knockdowns using three target sgRNAs and a scrambled sgRNA control. CD133⁺ Caco-2 colon cancer cells and 1205Lu cells served as positive and negative controls, respectively. Images were taken at 10x magnification. RT-PCR (b) and immunoblot analysis (c) show depletion of CD133 RNA and protein in BAK-R T3 compared to BAK-R SC or BAK-R cells. (d) Transwell cell invasion assays showed decreased cell invasion in CD133-depleted BAK-R T3 cells, compared to BAK-R, control SC sgRNA, or T1 and T2 cells. (e) Injection of zebrafish with a 1:1 mixture of BAK-R SC (red) and BAK-R CD133 knockdown T3 cells (green) and representative images after 5 days; (f) quantification of % metastasis to the zebrafish tail. *, **, *** represent p < 0.05 and p < 0.01, and p < 0.001, respectively.