HA-RAL Synergy in Skin Figure 5. The synergistic effect of RAL and HAFi in the protein expression of pro-HB-EGF in mouse skin and in human skin atrophy. A. Western blot analysis on the protein extracts of vehicle-, RAL-, HAFi- or RAL- and HAFi-treated SKH1 hairless mice for active HB-EGF (,15 kDa) (a), pro-HB-EGF (,25 kDa) (b) and erbB1 (,170 kDa) (c) and loading control a-tubulin (50 kDa) (d). B. RAL and HAFi show synergy in the correction of dermatoporosis. Skin thickness in untreated young, untreated atrophic aged, HAFi alone-treated atrophic aged or RAL and HAFi-treated atrophic aged human skin measured by echography. The results are presented as box-plots with median value (m). p = 0.001 (young untreated versus atrophic aged untreated); p,0.01 (atrophic aged untreated versus atrophic aged treated with HAFi); p,0.001 (atrophic aged untreated versus atrophic aged treated with RAL and HAFi); p,0.05 (atrophic aged treated with HAFi versus atrophic aged treated with RAL and HAFi) (nonparametric Mann-Whitney U test). C. Clinical view of atrophic human forearm skin before (a) and 1 month after topical treatment with RAL and HAFi (b). Note the decrease of wrinkles, hematoma and pseudoscars, and smoothening of the skin after RAL and HAFi treatment. Histological view of atrophic human forearm skin before (c) and 1 month after topical treatment with RAL and HAFi (d). Note the significant epidermal hyperplasia, decrease of elastosis and increase of collagen in the dermis after RAL and HAFi treatment. doi:10.1371/journal.pone.0014372.g005 Samples were loaded in nonreducing SDS sample buffer, subjected to electrophoresis, and transblotted onto 0.45 mm pore- size nitrocellulose membrane. Antibodies used for Western blot analysis were anti-CD44 standard (Bender MedSytems); anti- CD44v3 (Bender MedSystems); anti-MMP-7 (G-20) (Santa Cruz Biotechnology); anti-pro-HB-EGF (M-18) (Santa Cruz Biotech- nology); anti-HB-EGF neutralizing antibody (R&D Systems, Minneapolis, MN); and anti-erbB1 and anti-erbB4 (Santa Cruz Biotechnology). In vitro keratinocyte proliferation assay Epidermal keratinocytes were isolated and cultured in 96-well plates (Becton Dickinson) as described previously [16]. On day 2 of culture, HAFi (100 mg/ml), RAL (2 mM), RA (2 mM), ROL (2 mM), ROLP (2 mM), 4-MU (0.6 mM) (Sigma), hyaluronidase (1 U/ml) (Sigma), RAL+HAFi, RAL+HAFi, RA+HAFi, ROL+ HAFi or ROLP+HAFi, monoclonal anti-human amphiregulin (AR) neutralizing antibody (100 ng/ml), monoclonal anti-human erbB1 neutralizing antibody (isotype IgG1) (100 ng/ml), mouse recombinant TIMP-3 (100 ng/ml) or human HB-EGF (5 ng/ml) (R&D Systems), TPA (1 ng/ml) (Sigma), human EGF (50 ng/ml) (Sigma), in the presence or absence of anti-HB-EGF neutralizing antibody (10 ng/ml) (R&D Systems), was added to the cultures. Mouse IgG1 was used as a control of anti-erbB1. 48 hrs later 1 mCi of [3H]thymidine (Amersham, Buckinghamshire, UK) was added to each well. Isotope incorporation was evaluated 24 hrs later in a Beckman LS 1801 b counter (Beckman Coulter, Fullerton, California). All experiments were done in triplicate and repeated 5 times, and the results were expressed as the mean of incorporated counts per minute for each condition tested. PLoS ONE | www.plosone.org 7 December 2010 | Volume 5 | Issue 12 | e14372