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

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

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

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