After UVB treatment NRP1-deficient keratinocytes show a stronger activation of p53, as compared to control cells.

<p>Following UVB irradiation of keratinocytes in vitro, the expression levels of the pro-apoptotic factors puma and noxa showed a comparable increase in control and NRP1-deficient cells (A, B). In contrast to this, NRP1-knock out keratinocytes are characterized by a stronger and more persistent activation of p53, as reflected by increased phosphorylation of serin residue 20 (C). Quantitative RT-PCR and Western blotting was repeated independently with cells isolated from at least three different animals. The relative expression indicates the fold-change in comparison to the non-irradiated control lane. Significant changes (analysis of 3 blots, p<0.05) are indicated by an asterisk.</p

Expression and epidermis-specific deletion of neuropilin 1 in mice.

<p>Immunofluorescent staining for NRP1 in cryosections of normal human skin (A) or skin from 8 weeks old mice (B) shows protein expression in all layers of the epidermis, with a tendency towards stronger staining in the suprabasal layers (green fluorescence, nuclear stain in red with propidium iodide). For conditional keratinocyte-specific ablation of NRP1 expression, Keratin 14-Cre mice were crossed with mice homozygous for a mutated NRP1 gene containing loxP sites flanking the exon 2 (C). Genotyping of animals was performed by PCR (D). Keratinocytes isolated from newborn mice were checked by quantitative real time PCR for NRP1 mRNA levels (E). Additionally, NRP1 expression in the epidermis was assessed by immunofluorescent staining and demonstrated absence of detectable NRP1 protein in the epidermis of NRP1epi−/− mice (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050944#pone-0050944-g001" target="_blank">Figure 1 F, G</a>, green fluorescence, the broken line reflects the epidermal basement membrane).</p

UVB irradiation leads to increased apoptosis in NRP1-deficient epidermis in vivo.

<p>To investigate UVB-induced apoptosis in vivo epidermis-specific NRP1 knock out mice and control animals were irradiated with 1 J/cm². 24 hours later skin biopsies were stained for active caspase 3 in control (A) or knock out (B) animals. Apoptotic cells were counted and analyzed for statistically significant differences (C). Additionally, apoptotic cells were stained by TUNEL assays (D, E) and cells were quantified (F). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050944#pone-0050944-g004" target="_blank">Figure 4</a> (A to F) shows one representative example out of three independent experiments.</p

Induction of apoptosis by UVB irradiation in NRP1-deficient keratinocytes and epidermis.

<p>Primary murine keratinocytes were isolated from epidermis-specific NRP1-deficient or control animals irradiated with UVB light (20 mJ/cm²) in vitro. 24 h after irradiation keratinocytes were harvested and stained with popidium iodide for cell cycle analysis by FACS (A–D). Cells in sub G1 phase were considered to be apoptotic. After UVB treatment 20.7% of NRP1-deficient keratinocytes were apoptotic, whereas only 10.9% of control cells were in subG1 phase. The figure shows one representative example out of three independent experiments.</p

Differential regulation of Bcl-2 in NRP1-deficient and control keratinocytes following UVB irradiation.

<p>24 h after UVB irradition protein extracts from mouse epidermis and from primary keratinocytes cultured in vitro were prepared and analyzed by Western blotting. Prior to UVB treatment the amounts of Bcl-2 are comparable between control and NRP1-deficient samples (A). After UVB application Bcl-2 levels decrease significantly (p<0.05, indicated by an asterisk) in NRP1-deficient epidermis and cells, whereas control tissue and cell extracts show no significant changes in Bcl-2 amounts. No significant changes were observed in the levels of Mcl-1, phospho-p38 (only the induction 1 hour after UVB was significant, but present in control and NRP1-deficient cells) and phospho-Erk (B, C). All Western blots were repeated independently with extracts from tissue or cells from at least three different animals. The relative expression expresses the fold-change in comparison to the non-irradiated control lane.</p

NRP1-deficient keratinocytes are characterized by a lack of Rac1 activation after irradiation with UVB light.

<p>Like in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050944#pone-0050944-g005" target="_blank">Figure 5</a>, keratinocytes or animals were subjected to UVB treatment and protein extracts from mouse epidermis or from primary keratinocytes cultured in vitro were prepared after 24 h and analyzed by Western blotting. Compared to control keratinocytes, phospho-Akt activation is longer persistent in NRP1-deficient cells (A). Additionally, NRP1-knock out keratinocytes did not activate Rac1 after UVB irradiation (B). All Western blots were repeated independently with extracts from tissue or cells from at least three different animals. The relative expression expresses the fold-change in comparison to the non-irradiated control lane.</p

Microscopic characterization of keratinocyte-specific NRP1-deficient mice.

<p>Hematoxylin/eosin staining shows no differences in skin architecture between control and NRP1-deficient mice postnatally or in adult mice. Likewise, no changes in the expression of various differentiation markers could be detected. These markers included keratin 14 for basal keratinocytes, keratin 10 for suprabasal keratinocytes and loricrin for late stage differentiation, preferentially in the granular layer.</p