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0001% nile red in glycerin/water (75/25, v/v), as described previously (18). Biopsies were postfixed with either 0.25% ruthenium tetroxide or 1% aqueous osmium tetroxide (3). Ultrathin sections were examined in a Zeiss electron microscope (Jena, Germany) (3). Images were captured using Digital Micrograph 3.10.0 software (Gatan, Inc., Pleasanton, CA, USA). Density of lamellar bodies in the first outmost layer of stratum granulosum was quantitated per 4-cm2 area from electron micrographs. mRNA expression was measured by real-time quantitative PCR (QPCR). Lipid synthetic enzymes [serine�Cpalmitoyl transferase (SPT) and fatty acid 2 hydroxlase (FA2H)] and lipid transporters [ATP-binding cassette A12 (ABCA12)], CAMP, and mBD3 expression were assessed as described earlier (19,20). Primer sequences are in Table?S1. Expression of mRNAs was normalized to GAPDH. Results for in vivo studies are presented as the percentage Resminostat of vehicle-treated control (vehicle as 100%) while results for in vitro studies are presented as the ratio to untreated controls. Data are expressed as the means?��?SEM. Unpaired two-tailed Student t-test with Welch��s correction was used to determine significant differences when two groups were compared. One-way ANOVA with post-Tukey test was used when more than three groups were Smad inhibitor compared. We first assessed whether the topical CHM improves epidermal permeability barrier function in normal murine skin after topical applications of CHM twice daily for 7?days. No changes in gross or histological appearance were apparent (not shown). Although basal transepidermal water loss (TEWL), surface pH, AMPK inhibitor and SC hydration remained unchanged in CHM- versus vehicle-treated groups (Fig.?S1a�Cc), barrier recovery significantly accelerated in CHM-treated mice after repeated tape stripping (Fig.?S1d). Because both epidermal lamellar body (LB) formation and secretion are crucial for barrier function (18), we next assessed whether these parameters change following topical CHM treatment. Premature secretion of LB was evident, and LB density increased before and 2?h after acute barrier disruption in CHM-treated skin (Fig.?1a�Cd). Quantitative studies in coded, randomized micrographs confirmed that a significant increase in LB density occurred in the cytosol of stratum granulosum cells following CHM treatment (CHM-treated 7.88?��?1.03 versus vehicle-treated 4.58?��?0.87, P?
