FGF22 in Skin Development and Repair Figure 6. FGF signaling in HaCaT cells. A) HaCaT keratinocytes that had been serum-starved overnight were stimulated with FGF7, FGF10 or FGF22 (100 ng/ml) for 0, 15, 30 or 60 minutes and lysed in sample buffer prior to Western blotting. FGF7 and FGF10 elicited rapid ERK phosphorylation, whereas FGF22 yielded no significant response (phospho-ERK staining appears stronger due to increased length of exposure – to allow visualization of bands). B and C) Serum-starved HaCaT cells were stimulated as above with 100 ng/ml FGF7 (B) or FGF10 (C) either following a 4 h pre-treatment with FGF22 (100 ng/ml) or concomitant with FGF22 treatment. In each case, experiments were repeated (n = 5) and bands quantified using ImageJ (bars represent SEM). No reduction in ERK signalling was observed in either case. D) Forty-eight hours post-transfection with scrambled control non-targeting siRNA or FGF22 siRNA (both at 10 nM), serum-starved HaCaT cells were stimulated with either FGF7 or FGF10 (both 100 ng/ml) for 15 or 60 minutes and levels of phospho-ERK and total ERK analysed by Western blot as described above. No difference in ERK phosphorylation was detected in either case, with experiments repeated (n = 3) and bands quantified using Image (bars represent SEM). doi:10.1371/journal.pone.0039436.g006 Genotyping Genomic DNA isolated from ear snips or tail biopsies was used for PCR genotyping. Primers for the wild type (59-TTGTGAAC- CAAGATGGCAGG-39 and 59-CCAGCTTTGGACTT- CATCTG-39) and knockout (59-TCGACTAGAGGAT- CAGCTTG-39 and 59 CAGGCCAGCATAGTCTACTT-39) fgf22 alleles generated 286 bp and 130 bp PCR products, respectively. RT-PCR RT-PCR was performed to confirm FGF22 transcript deletion in fgf22 knockout mice. Total RNA was isolated from brains of adult wild type, heterozygous and knockout animals using TRI reagent (Sigma, Poole, UK), and reverse transcribed (Super- ScriptTM II RT, Invitrogen, Paisley, UK) using random hexamer primers. RT-PCR was performed using primers (59-ATTCTA- GAATGCGCAGCCGCCTCTGG-39 and 59-ATGGGCCCT- CAAGACGAGACCAAGAC-39) that amplified a 482 bp frag- ment of the FGF22 cDNA. GAPDH was amplified as a control housekeeping gene using published primers for mouse and human sequence [19,35]. Realtime RT-PCR Realtime RT-PCR was performed to assess relative mRNA expression levels of Fgf7 and Fgf10 in Fgf22 wild type and knockout mice. RNA extraction and reverse transcription was performed as above. Similarly, expression levels of FGF22 and NRF2 were quantified in HaCaT cells treated with scrambled control or FGF22 targeting siRNA pools. RNA was isolated using TRI reagent according to the manufacturer’s instructions and as for skin samples. Primers used were Fgf7 (59-GAACAAAAGT- CAAGGAGCAACC-39 and 59-GTCATGGGCCTCCTCC- TATT-39), Fgf10 (59-GAGAAGAACGGCAAGGTCAG-39 and 59-CTCTCCTGGGAGCTCCTTTT-39) and NRF2 [30]. FGF22 primers were as for RT-PCR. All reactions were performed in triplicate. A negative control was performed with all the mix components except the cDNA sample. GAPDH primers were used as internal control. The reaction was performed using SYBR green (Qiagen, Crawley, UK) in a StepOne Plus Realtime PCR System (Applied Biosystems, Life technologies, USA) thermal cycler according to manufacturer’s instructions. The results were evaluated using the 22DDct method [36]. Histological Analysis Normal and wounded back skin and tail skin were harvested and processed as described previously (Grose et al., 2007). Sections (4 mm) were analyzed using Haematoxylin/Eosin, anti-59bromo- deoxyuridine (BrdU) and Sirius red staining. Hair Analysis Hair subtypes were analyzed by plucking hairs from the middle of the back of five eight weeks old wild type and fgf22 knockout female mice (n = 100 hairs per genotype). They were measured and classified as guard, awl or auchene based on their appearance [26] under a light microscope (Axiophot, Zeiss, Welwyn, UK). Immunohistochemistry For detection of proliferating cells labelled with BrdU, paraffin sections (4 mm) were incubated with a peroxidase-conjugated monoclonal antibody directed against BrdU (1:500 dilution; Abcam, Cambridge, UK) for 1 h at room temperature. The signal was amplified using a StreptABComplex/HRP kit (Dako, Ely, UK) according to the manufacturer’s instructions, before peroxidase detection with a 3,39- Diaminobenzidine substrate kit (Dako). Sections were counterstained with Mayer’s haemalum and mounted with Permount (Fisher Scientific, Loughborough, UK). BrdU Scoring Cells exhibiting brown nuclear staining or showing mitotic figures were included in deriving the proliferation score. Cells stained in any of the categories 1 (faint), 2 (moderate) and 3 (strong) were considered positive whereas cells showing an absence of brown nuclear staining were considered negative. The percentage positive BrdU score was calculated by counting the total number of positive and negative cells over a specified field and then deriving the percentage of positive cells. A field was defined as the grid area provided in the microscope graticule (10610 squares) under a x400 magnification (10x ocular and 40x objective). Ten fields per sample were counted. The fields to be included in the score were chosen on the basis of the observer’s interpretation of fields best representing the heterogeneity of wound tissue across the sample. Sirius Red Staining To evaluate collagen organization in healing wounds, paraffin sections of full thickness 14 day wounds of fgf22 wild type and knockout mice were stained with Sirius red. In bright-field microscopy, Sirius red stains collagen fibres in red on a pale yellow background. The same slides, when examined through a microscope with cross-polarised light show that the larger collagen fibres (collagen type I) are bright yellow or orange, and the thinner ones, including reticular fibres and collagen type III, are green. The more mature and well organized the collagen fibres, the greater the birefringence intensity. De-waxed and hydrated paraffin wound sections were stained in Picro-sirius red (0.1% Sirius Red F3B in saturated aqueous solution of Picric Acid) for one hour, then washed in two changes of acidified water (0.5% Glacial Acetic Acid), dehydrated and mounted as above. Tail Epidermis Wholemounts Epidermal wholemounts were prepared by peeling tail skin off the bone, after making a scalpel incision along the length of the tail, and cutting into 5 mm2 pieces. These were incubated in 5 mM EDTA in PBS for 4 h at 37uC. Epidermal sheets were peeled away from the dermis and fixed in 1% Acetic Acid/95% overnight at 4uC. After rinsing in 100% ethanol, specimens were PLoS ONE | www.plosone.org 9 June 2012 | Volume 7 | Issue 6 | e39436