RORα-induced keratinocyte differentiation is partially mediated by FOXN1.

<p>(<b>A–C</b>) HKCs transfected with control siRNA or siRNA against FOXN1 were infected with retroviruses expressing GFP or RORα4 24 hours later. Samples were collected after additional 72 hours for qRT-PCR or western blot analysis of the indicated genes. qRT-PCR data are presented as mean fold-change over controls ± S.E.M. ***p<0.001, **p<0.01, *p<0.05, N = 3.</p

Increased expression of RORα4 triggers differentiation and inhibits proliferation of HKCs.

<p>(<b>A</b>) Real time qRT-PCR and western blot analysis of differentiation markers in HKCs at 72 h after infection with retroviruses expressing RORα4 or GFP alone. The mRNA level of each gene is normalized for 36B4 expression, and presented as mean fold-change over control ± S.E.M., ***, p<0.001, N = 3. (<b>B</b>) RT-PCR analysis of the indicated genes in HKCs under growing versus differentiating conditions as induced by suspension culture for 24 hours. Results are presented as mean fold-changes over control ± S.E.M., ***, p<0.001, N = 3. (<b>C–D</b>) Real time qRT-PCR analysis of the indicated genes in samples from (A). Data are presented as mean fold-change over controls ± S.E.M., ***, p<0.001, N = 3. (<b>E</b>) Alamar blue cell proliferation assay of HKCs in response to elevated RORα4 expression. Data are presented as mean fold-change of fluorescence intensity ± S.E.M. over control cells at day 2 after infection. **, p<0.01, N = 3. (<b>F</b>) Clonogenicity assay of HKCs in response to increased RORα4 expression. HKCs expressing RORα4 or GFP alone were analyzed for colony formation after 9 days of culture. Data are presented as mean percentage-change ± S.E.M. over control cells. ***, p<0.001, N = 3.</p

Silencing of RORα expression inhibits keratinocyte differentiation.

<p>(<b>A</b>) Real time RT-PCR and western blot expression analysis of the expression of indicated genes in HKCs transfected with two separate siRNAs against all RORα isoforms or control siRNA. Cells were harvested at day 4 after transfection when they reached 100% confluence. mRNA levels were normalized for 36B4, and presented as mean fold-change over control ± S.E.M. ***, p<0.001, N = 3. (<b>B</b>) Real time qRT-PCR and western blot analysis of indicated gene products in HKCs infected with lentiviruses expressing control shRNA or an shRNA against RORα. Cells were harvested 4 days after infection. Values are presented as mean fold-change over control ± S.E.M. **, p<0.01, ***, p<0.001, N = 3. (<b>C</b>) Western blot analysis of loricrin and filaggrin expression in HKCs transfected with control or RORα siRNAs. Seventy-two hours after transfection, sub-confluent cells were re-plated onto poly-HEMA coated plates, and harvested after 20 h culture in suspension. (<b>D</b>) Real time qRT-PCR analysis of the indicated lipid/epidermal barrier related genes in HKCs plus/minus RORα knockdown as in (C). Values are presented as mean fold-change over control ± S.E.M. **, p<0.01, ***, p<0.001, N = 3.</p

RORα positively regulates the expression of FOXN1.

<p>(<b>A–B</b>) HKCs with increased (A) or knocked-down (B) RORα expression were analyzed for expression of mature FOXN1 and Notch1 mRNA by real time qRT-PCR. Values are presented as mean fold-change over control ± S.E.M., ***, p<0.001, N = 3. (<b>C</b>) Chromatin immuno-precipitation (ChIP) analysis of RORα binding to the regulatory region of the FOXN1 gene. Mat-inspector software was used to search for RORα response elements (ROREs) within –6 kb and +2 kb from the transcription starting site (TSS) [Top]. Human epidermis was processed for ChIP assays utilizing rabbit antibodies specific for RORα or non-immune IgG control followed by PCR amplification of the indicated regulatory regions of the FOXN1 gene. The relative amount of precipitated DNA was calculated after normalization for total input chromatin, according to the following formula <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070392#pone.0070392-Frank1" target="_blank">[59]</a>: % total = 2^ΔCt×5, where ΔCt = Ct (input) – Ct (immunoprecipitation), Ct, cycle threshold. Statistical significance of the results was determined by unpaired Student’s <i>t</i>-test, comparing the ratio RORα/IgG signal for each binding site relative to the one for the binding site at the RORE negative region. **, p<0.01, N = 3. (<b>D</b>) HKCs with increased or knocked down RORα expression were analyzed by qRT-PCR for primary FOXN1 transcript levels, using a primer corresponding to the first intron/exon junction. Samples were the same as described in (A–B). Values are presented as mean fold-change over control ± S.E.M., ***, p<0.001, N = 3.</p

p21-mediated down-regulation of genes involved in mitotic control in human keratinocytes.

<p>A. Primary human keratinocytes were infected with adenovirus expressing p21 and ad-GFP as a control. 24 h after infection total RNA was prepared and the mRNA levels of the indicated genes were analysed by RT-qPCR. The values are means ±S.E. from three determinations. B. keratinocytes were transfected with siRNA for p21 and a negative control siRNA. Total RNA was prepared and the mRNA levels of the indicated genes were analysed by RT-qPCR. C. Primary human keratinocytes were transfected with p21 siRNA or control siRNA. 48 h after lipofection total RNA was prepared and the mRNA levels of the indicated genes were analysed by RT-qPCR.</p

p21-mediated repression of genes is not dependent on CDK2.

<p>A. K562 cells were transiently transfected with a short-hairpin CDK2 (“shCDK2”) vector and the empty vector (pLKO.1, “Vo”). 24 h after transfection the cells were treated with 75 µM ZnSO₄ for 12 h to induce p21 and the silencing of CDK2 was assayed at the protein level by immunoblot (left panel) and at the mRNA level by RT-qPCR (right panel) The expression of p21 and actin was also determined to control the p21 induction by Zn²⁺ and the protein level, respectively. B. The expression of p21 was induced with 75 µM ZnSO₄ in K562 transfected with sh-CDK2 and 12 h later, total RNA was prepared and expression of the indicated genes was determined by RT-qPCR. The values are means ±S.E.M. from two independent experiments and two determinations for each RNA.</p

p21-mediated the down-regulation of genes involved in cell cycle.

<p>A.The expression of p21 was induced in Kp21-4 cells by 75 µM ZnSO₄ and 3, 6 and 12 h later, total RNA was prepared and expression of the indicated genes was determined by RT-qPCR. In some cases an alternative name is given into brackets. Cyc., cyclin. The values are means ±S.E.M. from two independent experiments and two determinations for each RNA. B. Kp21-4 cells were treated for 6 h with 75 µM ZnSO₄ to induce p21. Chromatin immunoprecipitation was carried out with anti-histone H3 and anti-acetylated-histone H3 antibodies and (as specificity control) rabbit IgG. The DNA in the immunoprecipitated chromatin was measured by quantitative PCR. The amplicons encompass the transcription start site of the indicated genes. A regulatory sequence of the promoter of rDNA was used as a control. The results are expressed as the ratio of DNA enrichment in chromatin immunoprecipitated with anti-H3 versus acetylated-H3 (“Ac-H3”) and normalized to the values obtained in uninduced cells. The values are the means ±S.E.M of two independent ChIP experiments with two PCR determinations each.</p

p21 represses the activity of human CCNE2 promoter.

<p>A. K562 cells were transfected with luciferase reporters carrying the promoters cyclin E1 and E2 genes (<i>CCNE1</i> and <i>CCNE2</i>), along with an expression vector for p21 and the corresponding empty vector, and a beta-gal plasmid for transfection efficiency normalization. 24 h after transfection the luciferase activities were determined. The data are normalized to the activity of cells transfected with the empty vector. Lower panel: immunoblot analysis of the transfected cells to assess the expression of p21. B. Kp21-4 cells were transfected with luciferase reporters carrying the promoters cyclin E1 and E2 genes as in (A) and 24 h later the cells were treated with 75 µM ZnSO₄ and after 24 h the luciferase activities were determined. The data are normalized to the activity of cells without ZnSO₄. Lower panel: immunoblot analysis of the transfected cells to assess the expression of p21.</p

Genes regulated by p21 in human keratinocytes related to cell cycle and cell division.

<p>A. Primary human keratinocytes were infected with recombinant adenoviruses expressing p21 (Ad-p21), p27 (Ad-p27). As a control, cells were also infected with adenovirus expressing GFP (Ad-GFP). The mRNA expression of p21 (upper graph) and p27 (lower graph) was determined by RT-qPCR 24 h after infection. Data are represented relative to the expression in Ad-GFP-infected cell. B. Heat-map showing the 82 genes changed by p21 related to cell cycle and cell division with an expression change > 2.3-fold. The names of the regulated genes are indicated at the right. Green indicates genes down-regulated by p21 and red genes up-regulated.</p

The N-terminal region of p21 is required for its effect as gene repressor.

<p>A. Schematic representation of the p21 constructs used. The proteins carry the green fluorescent protein (GFP) in the N-terminal. The position of the CDK binding domain (CDB) and the PCNA binding domain (PCNA) are indicated. B. K562 cells were transfected with expression vectors for the full-length p21 protein (FL), the p21 amino-terminal region (NT) and the p21 carboxy-terminal region (CT). 24 h after transfection the cells were sorted by flow cytometry and the expression of the indicated genes was analysed by RT-qPCR. The values are means ±S.E. from two transfections and two determinations of each mRNA.</p