Fig. 2

The reduction in NAMPT expression decreases tumorigenesis in HNSCC cell lines. A Measurement of NAMPT expression levels in tumorspheres and total extract by RT‒qPCR. B Measurement of CD10, CD184, CD19, CD133 and CD166 expression levels in NAMPT-overexpressing and EV cells by RT‒qPCR. Control cells (parental + vector only in black; overexpressing NAMPT in orange). Further validation of NAMPT overexpression is shown in Supplementary Fig. 1. C) WB showing reduced expression of NAMPT in NAMPT CRISPR clones, which were 2 different clones for each cell line. D Reduced levels of NAD + and NAD-total pools in NAMPT CRISPR clones with respect to parental cultures. E Reduced clonability in NAMPT CRISPR clones with respect to parental cultures. Representative images of the clones are shown. F Competition assay of control and NAMPT-CRISPR-GFP cells. Equal numbers of cells were seeded in the same plate, and 30 days later the GFP-cell percentage was quantified by FACS. NAMPT CRISPR clones have a lower ability to compete against parental cultures (with higher NAMPT expression) in normal culture media. Only clone D11 showed a competition capacity similar to that of the parental cells. G Tumor growth and tumor size of tumors from parental or NAMPT CRISPR clones. Tumor growth in vivo in xenografts (N = 4) from NAMPT CRISPRs and control cells. Representative images of tumor size are shown. The mean ± standard deviation is presented. Statistical analysis was performed with Student’s t test (*p < 0.05; **p < 0.01; ***p < 0.001)