Fig 2: Stem properties of CD10, CD184, CD19, CD133 and CD166 subpopulations in HNSCC cell lines. A) Measurement of CD10, CD184, CD19, CD133 and CD166 expression levels by RT‒qPCR in tumorspheres (TO) and total extracts (TE) of RPMI-2650 and Detroit-562 cells. B) Tumorsphere assay of positive and negative subpopulations isolated by cytometer. C) Tumor growth in vivo in xenografts (N = 6) from positive and negative subpopulations isolated by a cytometer. 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). ND: not determined. D) Cellular plasticity of the different subpopulations in HNSCC. Analysis of the positive cells for CD10, CD184, CD19, CD133 and CD166 markers by FACS of the isolated positive and negative subpopulations after being separated by cytometry and cultured for 1 or 2 weeks depending on the cell line. Non selected cells were used as a control. ND: not determined

Fig 3: 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)

Fig 4: Effect of WDR66 up/downregulation on stem cell phenotype in head and neck cancer cell lines. (A) Tumorsphere formation assay in head and neck cancer cells with up/downregulated WDR66 and control cells. The result is expressed in % tumorspheres because the control has been normalized to 100%. Below is the area in mm2 of the formed tumorspheres. Representative images of the tumorspheres formed in the two cell lines. (B) Study of the levels of proteins related to the stem cell phenotype by Western blot in cells with up/downregulation of WDR66 and control cells. (C) Quantification by flow cytometry (FACS) of the expression of the surface markers CD184 and CD10 in the up/dowregulation models. The mean of 3 independent experiments ± SEM is presented. Statistical analysis was performed with Student's t test (*p<0.05; **p<0.01; ***p<0.001). The absence of an asterisk means that the data are not statistically significant. EV=Empty vector; WDR66= WDR66 overexpressing cells.