Role of RAB27A in Skin Color Determination Figure 3. The ratio of keratinocytes with incorporated melanosomes is significantly higher when co-cultured with melanocytes expressing higher RAB27A amounts. (A) Melanocytes with higher RAB27A expression numbered 1, 4 and 6 and cells with its lower expression numbered 8, 16, 17 and 18 as explained in Figure 2 are divided into High and Low groups. Immunohistochemistry for melanosomal protein gp100 (green) in co-culture using High (B) or Low melanocytes (C) is demonstrated. Red arrows indicate melanosomes transferred into keratinocytes. Nuclei are stained blue with DAPI. Scale bars = 10 mm. (D) The ratio of keratinocytes harboring transferred melanosomes is compared when co-cultured with High and Low melanocytes. The values represent means 6 SD. Statistical significance was evaluated by the Student’s t-test, and a p value,0.05 is considered statistically significant. doi:10.1371/journal.pone.0041160.g003 keratinocytes co-cultured with melanocytes treated with the RAB27A shRNA-lentivirus compared to those co-cultured of the SCR-lentivirus treated cells (Fig. 5F). Following the evaluation with the co-culture system, HSSs in vivo were constructed using RAB27A-shRNA or SCR-lentivirus treated melanocytes for further investigation. Four weeks after the grafting of the cell mixture, the surface color of reconstructed HSSs composed of RAB27A shRNA-lentivirus treated melanocytes was lighter compared to those composed of melanocytes treated with the SCR-lentivirus (Fig. 6A, 6B). Immunostaining using an antibody specific for the melanosomal protein tyrosinase-related protein-1 and RT-PCR with a probe specific for RAB27A confirmed the melanosome aggregation within melanocytes and the reduced expression of RAB27A mRNA, respectively, in the epidermis when melanocytes treated with the lentiviral vector expressing shRNA for RAB27A were used (Fig. 6C–E). Consistently, HSSs PLoS ONE | www.plosone.org 5 July 2012 | Volume 7 | Issue 7 | e41160