HA-RAL Synergy in Skin Figure 3. RAL and HAFi have a synergistic effect on the protein and mRNA expression of CD44 in mouse skin. A. Immunostaining of sections of vehicle- (a), RAL- (b), HAFi- (c) or RAL- and HAFi- (d) treated DBA/1 mouse dorsal skin with anti-CD44 antibodies. Note the hyperplasia and increase in diffuse CD44 expression in the epidermis in RAL-, HAFi- and RAL- and HAFi-treated mice. B. Immunostaining of sections of vehicle- (a), RAL- (b), HAFi- (c) or RAL- and HAFi- (d) treated DBA/1 mouse dorsal skin with anti-CD44v3 antibodies. Note the hyperplasia and increase in suprabasal CD44v3 expression in the epidermis in RAL-, HAFi- and RAL- and HAFi-treated mice. C. Western blot analysis for CD44 (,90 kDa) performed on the epidermal proteins extracted from the RAL-, HAFi- and RAL- and HAFi-treated skin of SKH1 hairless mice. a-tubulin (50 kDa) has been used as a loading control. D. Northern blot analysis of CD44 RNA expression in RAL-, HAFi- and RAL- and HAFi-treated skin of DBA/1 mice. Note the increase of the three CD44 transcripts (5.2, 4.0 and 3.2 kb) in RAL-, HAFi- and RAL- and HAFi-treated skin. doi:10.1371/journal.pone.0014372.g003 using a Braun sonifier for 30 min at 400 W on ice. The fragments were then separated on a Sephacryl S-400 HR 4x80 cm size exclu- sion gel filtration column (Pharmacia Diagnostics, Uppsala, Sweden). The column was calibrated using Pullulan standards (Showa-Denko, Kawasaki, Japan) from 20’000 to 800’000 Da. 100 fractions of 10 ml each were collected from the column with a Frac. 100 fraction collector (Pharmacia) at a flow rate of 1 ml/min. The HA concentration of each sample was determined by a colorimetric dosage according to a BCA reducing sugar assay and an elution profile for the fragments was obtained. The fractions corresponding to the 50’000–400’000 Da were pooled, dialyzed against distilled water and lyophilized. The fragments were separated by 15% polyacrylamide gel (TBS) electrophoresis and visualized with an alcian blue and silver staining. HAFi were prepared in the form of cream in a neutral vehicle. Treatment of mice Groups of five adult (.3 month-old) SKH1 hairless, DBA/1 (The Jackson Laboratory, Bar Harbor, ME) or CD44-deficient (CD442/2) mice [14] were used. HAF, RAL or vehicle cream samples of 0.5 g were applied twice daily for 3 days to the dorsal skin of SKH1 hairless, DBA/1 or CD442/2 mice. The daily amount of RAL delivered corresponded to 250 mg. Animals were sacrificed two hours after the last application. All animals were handled in strict accordance with good animal practice as defined by the relevant national and/or local animal welfare bodies, and all animal work was approved by the Ethical Commission on Animal Experimentation of the University of Geneva and the Cantonal Veterinary Office of Geneva (ethics approval code 31.1.1035/2368/I). Treatment of healthy subjects and patients 7 healthy young adults (7 M) between 19 and 32 years (mean age 25.5 years), 3 patients with advanced age-related dermato- porosis (2 F; 1 M) between 60 and 88 years (mean age: 76 years) and 3 patients with dermatoporosis due to prolonged use of oral corticosteroids (3 F) between 74 and 86 years (mean age: 81 years) PLoS ONE | www.plosone.org 5 December 2010 | Volume 5 | Issue 12 | e14372