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. 2017 Aug 28;8(59):99514–99530. doi: 10.18632/oncotarget.20577

Figure 3. NAMPT expression increases tumorigenic and CIC properties.

Figure 3

(A, B) Q-RT-PCR shows NAMPT overexpression in either SF268 (A) or U251MG (B). (C, F) Western blot analysis shows NAMPT overexpression and NAMPT silencing with shRNA in SF268 (C) and U251MG (D). (E, F) Analysis of the growth curve indicates that NAMPT overexpression confers a proliferative advantage [*p < 0.05; **p < 0.01 with ANOVA compared to vector], whereas NAMPT underexpression slows proliferation [*p < 0.05, **p < 0.01 with ANOVA compared to vector]. (G, H) Clonogenicity assay results indicate that NAMPT overexpression increases the number of colonies [**p < 0.01 with ANOVA compared to vector], whereas NAMPT underexpression decreases the number of colonies [*p < 0.05 with ANOVA compared to vector]. (I) Tumorsphere-forming assay in both cell lines indicates that NAMPT overexpression increases both number and size, whereas NAMPT underexpression decreases tumorsphere number and size. (J, K) Analysis of clone phenotypes [holoclones – black, meroclones – gray, paraclones – white] shows that NAMPT overexpression increases the number of holoclones [**p < 0.01 with ANOVA compared to vector], whereas NAMPT underexpression decreases the holoclone number [*p < 0.05 with ANOVA compared to vector]. J shows data from SF268 cell line. K shows data from U251MG cell line. (L) CD133 analysis with FACS indicates that NAMPT overexpression increases CD133 levels [*p < 0.05 with ANOVA compared to vector], whereas NAMPT underexpression decreases CD133 levels [**p < 0.01 with ANOVA compared to vector]. (M) CD44 analysis with FACS indicates that NAMPT overexpression increases CD44 levels [**p < 0.01 with ANOVA compared to vector], whereas NAMPT underexpression decreases CD44 levels [*p < 0.05 with ANOVA compared to vector]. (N–Q) Single-cell Sphere-forming assay indicates that NAMPT overexpression increases both number and size, whereas NAMPT underexpression decreases tumorsphere number and size. (N) Single cell tumorsphere forming efficiency representative picture. (O) Single cell tumorsphere forming efficiency percentage. (Q) Single cell tumorsphere size. (P) Single cell colony (full culture) forming efficiency percentage.