Induction of K15 at high cell density.

<p>N-Terts were grown on coverslips at low (30%) or high (95%) cell densities in RM+. The cells were fixed in formaldehyde and immunostained with antibodies against K15 (a, b), K14 (c, d), involucrin (e, f) and cornifin (g, h). All slides were photographed at the same magnification. The insets in a and b were magnified in i and j, respectively, to show the presence of K15 aggregates, shown by arrows, in keratinocytes grown at low cell density (magnification bar = 20 µm).</p

Induction of K15 transcription in keratinocytes cultured at increasing cell densities.

<p>N-Terts growing in SFM at low (0.09 mM) (a) and high (1.8 mM) (b) calcium concentrations were allowed to reach the desired confluence before being used for expression of K1, K10, K14, K15, cornifin and FOXM1B. In (c) N-Terts at low calcium (0.09 mM) were plated at the required density, allowed to attach for 24 h, after which they were used to measure mRNA levels by qPCR. Each bar represents the mean±SEM where n = 3.</p

FOXM1B binding site on the K15 promoter.

<p>(a) K15 promoter divided into seven fragments (F1–F7) with the unique FOXM1B binding motif (K15) and its comparison with the consensus sequence (con) is shown and the nucleotides in red highlight the differences. (b) X-chromatin immunoprecipitation (X-ChIP): Primary human keratinocytes transduced with FOXM1B-GFP chimera were subjected to X-ChIP using FOXM1B and GAPDH (as control) antibodies. The immunoprecipitated DNA was quantified by qPCR using primers specific for different regions (F1–F7) (see panel a). All primers produced a single product (Melting peak) and the trace before (Total input) and after addition of FOXM1B (FOX) and GAPDH (GAP) antibodies (IP Fraction) is shown. Data for only 3 primer sets are shown for clarity. (c) The DNA fragments immunoprecipitated with FOXM1B antibody were quantified by qPCR. Each bar represents the mean±SEM where n = 3.</p

Knocking down PKCδ suppresses suspension-induced expression of K15.

<p>N-Terts were transfected separately with mock (no oligo), control siRNA or siRNA for PKCδ or PKCη. After 48 h of transfection, the cells were suspended in methylcellulose for 24 h, and used to determine the mRNA expression of K15, PKCδ and PKCη by qPCR. Each bar represents the mean±SEM where n = 3. (P<0.05, significant, *; P<0.01, very significant, **).</p

Influence of PKC activator and inhibitor on the expression of K15 in keratinocytes.

<p>(a) N-Terts in RM⁺ were exposed to different concentrations of PMA (0–500 nM) or DMSO for 24 h and mRNA expression was determined by qPCR. (b) The keratinocytes were exposed overnight to either 0 or 10 nM PMA, and protein levels were determined by western blotting. (c) N-Terts grown in SFM were suspended in 1.3% (w/v) methylcellulose with no additive, 0.02% DMSO, or 5 µM PKC inhibitor GF109203X for 24 h and the cells were used to determine mRNA expression. Freshly trypsinised single cell suspension of N-Terts was used as 0 h control cells. (d) N-Tert keratinocytes were suspended as in (c) with GF109203X and protein expression at 0 h or 24 h was determined by western blotting. Each bar represents the mean±SEM where n = 3. (P<0.01, very significant, **; P<0.001, extremely significant, ***). For immunostaining N-Terts growing on glass coverslips in RM+ were treated with 10 nM PMA (f, h, j, l) or DMSO control (e, g, i, k) for 24 h. The cells were fixed in 3.8% formaldehyde and immunostained with antibodies against K15 (e, f), K14 (g, h), involucrin (i, j) and cornifin (k, l) (magnification bar = 20 µm).</p

Induction of K15 in terminally differentiated N-Tert keratinocytes.

<p>N-Terts growing in SFM were suspended in DMEM +20% FCS containing 1.3% (w/v) methylcellulose. At 24 h the cells were harvested and washed with PBS before being suspended in 20–30 µl FCS. A 3–4 µl aliquot was streaked on glass slides. At the same time freshly trypsinised N-Terts were also streaked and used as 0 h control. The cells were dried in air, fixed in formaldehyde and immunostained with different monoclonal antibodies as described in the ‘Materials and Methods’ section. K15 (a, b), K14 (c, d), Involucrin (e, f), Cornifin (g, h). All slides were photographed at the same magnification. The cells in b, d, f and h were difficult to focus because they underwent aggregation in methylcellulose (magnification bar  = 20 µm).</p

<i>FOXM1</i> induces promoter hypermethylation of <i>p16^INK4A</i> gene in primary human oral keratinocytes.

<p>(<b>A</b>) Bisulfite modification and methylation specific absolute qPCR for the quantification of <i>p16^INK4A</i> promoter methylation status. Genomic DNA was first treated with sodium bisulfite prior to PCR pre-amplification of the promoter region of <i>p16^INK4A</i> (PCR^BS, 273 bp). Methylation specific (p16M-R/F) and methylation-independent (p16U-F/R) primers were then used to quantify the relative levels of methylated and unmethylated products within the PCR^BS sample using standard-curve based absolute qPCR method for each product, respectively. Melting analysis was performed to validate the qPCR specificity in detecting the two M and U products. (<b>B</b>) Bisulfite conversion and methylation specific qPCR were performed to measure the relative levels of unmethylated (U, melting temperature at 85.8°C) and methylated (M, 91.2°C) in either EGFP- or FOXM1-transduced primary NOK treated with either vehicle (DMSO) or 5Aza (1 µM, 3-day incubation with fresh drug replenishment daily). A total of n = 11 replicates from at least 4 independent experiments were performed. Statistical t-test significance notations *P<0.05 and ***P<0.001.</p

Upregulation of <i>FOXM1</i> (isoform B) induces a global shift in methylation pattern that mimics the cancer epigenome.

<p>(<b>A</b>) Genome-wide promoter microarray analysis of primary normal oral human keratinocytes expressing either <i>EGFP</i> (NOKG, black dots) or <i>FOXM1</i> (NOKF, yellow dots) and an established squamous cell carcinoma cell line (SCC15, red dots). Each dot represents a single gene. (<b>B</b>) A non-linear 2^nd order polynomial regression analyses were performed on the relative methylation patterns between NOKG vs NOKF (inverse correlation), NOKG vs SCC15 (inverse correlation) and NOKF vs SCC15 (positive correlation). (<b>C</b>) Gene selection criteria for differentially methylated genes between control (NOKG) and tests groups (NOKF and SCC15). 100-most hypermethylated and 100-most hypomethylated genes were inversely matched with differentially methylated genes from NOKF and SCC15. The adjacent gene lists show the shortlisted FOXM1-induced (also found in SCC15) differentially hypermethylated (red) and hypomethylated (green) genes compared to control NOKG cells. The CDKN2A (encodes <i>p16^INK4A</i>) gene, its promoter known to be hypermethylated in HNSCC, was included as a positive control for promoter hypermethylation. (<b>D</b>) Clinical tumour tissue sample correlation between the relative levels of methylation and gene expression of each shortlisted gene in a cohort of 10 patients with paired normal margin and HNSCC tumour tissue samples. Each dot represents mean ± SEM of each gene. Vertical error bars were derived from relative gene expression of 10 margin-tumour tissue pairs and horizontal error bars were derived from relative promoter methylation of 3 independent primary NOK (NOKG/NOKF) experiments. Correlation coefficient (R²) of a non-linear 2^nd order polynomial regression analyses were performed on all 30 candidate genes (left panel), 16 hypermethylated genes (middle panel) or 14 hypomethylated genes (right panel), respectively.</p

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.</p