Upregulation of FOXM1 suppressed <i>p16^INK4A</i> expression in primary human oral keratinocytes.

<p>(<b>A</b>) FOXM1 significantly supresses <i>p16^INK4A</i> mRNA and protein expression (inset figure) in primary normal human keratinocytes. GAPDH was used as a control for protein loading. Control cells (mock-transduced with empty retroviral particles or EGFP-transduced) did not show significant suppression of p16^INK4A expression. (<b>B</b>) Knockdown of a FOXM1-target gene <i>HELLS</i>, which regulates genome-wide methylation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034329#pone.0034329-Dennis1" target="_blank">[14]</a>, induced <i>p16^INK4A</i> and simultaneously suppressed <i>DNMT1</i> and <i>DNMT3B</i>, but not <i>DNMT3A</i> mRNA expression in a FOXM1-transformed malignant cell line (SVFN5) expressing constitutive levels of endogenous <i>HELLS </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034329#pone.0034329-Gemenetzidis1" target="_blank">[8]</a>. Each bar represents a mean ± SEM of triplicate transfection (48 h) with either siCTRL or siHELLS. *P<0.05, **P<0.01 and ***P<0.001 indicate the level of statistical significance compared to controls. (<b>C</b>) Endogenous <i>FOXM1</i> (isoform B) mRNA expression levels in 8 strains of primary human normal oral keratinocytes, 5 dysplastic and 11 HNSCC cell lines. Total <i>FOXM1</i> mRNA expression levels were measured in the EGFP and FOXM1-transduced NOK (NOKG and NOKF), respectively. (<b>D</b>–<b>J</b>) Third-order polynomial regression analyses were performed to obtain the R² coefficient of determination values which indicate the significance of co-expression between each gene with <i>FOXM1</i> across the 24 cell strains/lines indicated in panel C.