HA-RAL Synergy in Skin Figure 4. RAL and HAFi have a synergistic effect on the epidermal and dermal HA production in mouse skin. Epidermal (A) and dermal (B) HA content of vehicle-, RAL, HAFi- or RAL- and HAFi-treated skin of SKH1 hairless mice quantified by an enzyme-linked binding protein assay. The results are presented as the mean HA concentration 6 SEM of six animals per group. ***p,0.001 versus vehicle; **p,0.01 versus vehicle; *p,0.05 versus vehicle (student’s t test). doi:10.1371/journal.pone.0014372.g004 (for HAFi only treatment), and 3 patients with advanced age- related dermatoporosis (2 F; 1 M) between 75 and 92 years (mean age: 84 years) and 3 patients with dermatoporosis due to prolonged use of oral corticosteroids (2 F; 1 M) between 63 and 77 years (mean age: 71 years) (for RAL and HAFi treatment) were included in this study after obtaining written informed consent. Clinical studies were conducted with the authorization and according to the guidelines of Ethical Commission on Human Research of the University Hospital of Geneva. 0.05% RAL, 1% HAFi or vehicle cream samples of 0.5 g were applied twice daily for 3 months on the posterior side of the right or left arm, respectively. Histology Dorsal skin samples were fixed in 10% phosphate-buffered formaldehyde, embedded in paraffin, and processed for histolog- ical analysis. Sections were cut at 5 mm, mounted onto slides, and stained with hematoxylin-eosin according to standard procedures. Staining of skin sections for CD44, CD44v3, HABP, Ki-67 Paraffin-embedded sections (5 mm) were mounted onto slides, de-waxed in xylene, rehydrated in a graded ethanol series and prepared for immunoperoxidase staining according to standard procedures. Primary antibodies included: anti-CD44v3 (Bender MedSytems, Vienna, Austria; 1:100), anti-Ki-67 (Dako, Glostrup, Denmark; 1:20). After staining with the primary reagent for 1 hr at room temperature, sections were washed, incubated with biotinylated affinity-purified secondary antibody or with biotiny- lated anti-CD44 (PharMingen, San Diego, CA; 2.5 mg/ml) for 30 min at room temperature, washed, and treated with avidin- biotin-peroxidase for 30 min at room temperature. The sections were then washed with buffer and incubated in 0.05% DAB (3,39- diaminobenzidine; Sigma) and 0.03% H2O2 in phosphate buffer at room temperature. All sections were examined under a Zeiss axiophot microscope using appropriate filters. Epidermal and cutaneous thickness measurements Epidermal thickness of mice was measured by a graded ocular and multiplied by 10 to correct the scale. Cutaneous thickness measurements of the healthy subjects and patients were performed using a skin ultrasound system (Episcan, Longport Inc., Glen Mills, PA). Cell proliferation Epidermal and dermal Ki-67 positive cells were counted in ten fields per section at 406 magnification and the average value was calculated. RNA isolation and Northern blotting Total RNA from the back skin of SKH1 hairless, DBA/1 and CD442/2 mice topically treated with RAL was prepared using a TRIzol reagent kit (Invitrogen) and digested with RNase-free DNase 1 (Ambion). Eight micrograms of isolated RNAs were separated electrophoretically on a 1% agarose gel containing glyoxal and transferred onto a nitrocellulose membrane. The membrane was UV-cross-linked and probed with [32P]UTP-labeled hyaluronate synthase (HAS) cDNA (16106 cpm/ml). Hybridiza- tion with CD44 probe prepared using random primers and CD44 fragment purified from HindIII-EcoRI-digested mCD44H-pBS plasmid template (Kaya et al, 1997) was performed overnight at 60uC in 50% formamide, 16 sodium chloride-sodium citrate, 56 Denhardt’s reagent and 0.2% tRNA. The membrane was washed twice at 68uC for 30 minutes in 0.16 sodium chloride-sodium citrate and 0.1% sodium dodecyl sulfate. Kodak (Rochester, NY) X- Omat AR film was exposed overnight at 270uC. The integrity of total RNA was good, and the ratio of 28S and 18S ribosomal RNAs was ,2:1 in all samples. The hybridization signals were quantitated by scanning the autoradiograms with a laser densitometer and using the ImageQuant software. Western blot analysis Frozen mouse epidermis or dermis and human biopsy samples were incubated in extraction buffer containing 20 mM Tris/HCl (pH 7.5), 100 mM NaCl, 10 mM EDTA, 1% SDS, 10% glycerol and protease inhibitor cocktail (completeTM Boehringer, Biberach, Germany), minced, polytron-homogenized and sonicated on ice. Cell culture extracts were treated with the same buffer. Homogenates were spun in a microfuge for 20 min, and the soluble fraction was extracted and subjected to western blot analysis with appropriate antibodies. PLoS ONE | www.plosone.org 6 December 2010 | Volume 5 | Issue 12 | e14372