Figure 7. CEP55 and HELLS are putative FOXM1B target genes and are upregulated in both premalignant and HNSCC cell lines.

(A) mRNA levels of CEP55 and HELLS but not CENTD1 were significantly upregulated in SVFN transformed clones (solid bars) compared to non-transformed SVpgC2a parental wild-type cells (open bars). (B) Exogenous overexpression of FOXM1B (determined using isoform-specific qPCR, see Supplemental Fig S3), but not MCS (empty vector) or EGFP, in primary human normal oral keratinocytes (NHOK1) showed significant (***P<0.001) induction of endogenous mRNA of CEP55 and HELLS, respectively. Fold mRNA expression levels of FOXM1B (C), CEP55 (D) and HELLS (E) in a panel of oral premalignant cell lines (SVpgC2a, DOK, D19, D20, POE9n-hTERT, POE9n and OKT6) and HNSCC (CA1, UK1, CaLH2, CaLH3, CaDec11, CaDec12, H357, 5PT, PE3/JA, VB6, CaLH2-R, BICR31, SCC4, SCC9, SCC15 and SqCC/Y1) compared to the mean±SEM (n = 8) of normal primary human oral mucosa cells (NHOK1-5, 16, 376, 881). Insets show mean±SEM fold mRNA expression levels of normal (n = 8), oral premalignant (n = 7) and HNSCC cells (n = 16). All three genes show statistically significant (*P<0.05; **P<0.01; ***P<0.001) upregulation in both oral premalignant and HNSCC cell lines. (F) Linear regression analysis between FOXM1B and CEP55 expression in the panel of cell lines used in C and D. (G) Linear regression analysis between FOXM1B and HELLS expression in the panel of cell lines used in C and E. (H) Linear regression analysis between CEP55 and HELLS expression in the panel of cell lines used in D and E. Degree of correlation values (R²) are given in each panel as indicated. (I–J) ChIP-qPCR assays for promoters of CEP55 and HELLS in normal keratinocytes transduced with either Mock (empty virus), EGFP or FOXM1B. Antibodies for GAPDH and cMYC were used as controls for immunoprecipitiation. Representative qPCR curves and melting peaks of each ChIP fractions could be found in Supplemental Fig. S7.