PAX3 in Melanocytes Figure 2. Co-expression of PAX3 and MITF in melanocytic and melanoma cells. A–C) Double immunofluorescent staining showing co- expression of PAX3 and MITF in: (A) the epidermal melanocytes of normal skin; (B) primary melanoma; and (C) in an A2058 metastatic melanoma cell line. Arrowheads in (A) show PAX3-positive normal epidermal melanocytes. Lines in (A) and (B) demarcate epidermal-dermal border (EDB). The variable PAX3 expression was clearly visible in the A2058 cell line (C). For all these experiments depicted in the figure, PAX3 was labelled with rabbit polyclonal antibody (Invitrogen). D) Graph showing the overall number of MITF and PAX3 double labelled cells in normal skins, naevi, primary melanomas and melanoma metastases. Each column represents a percentage of MITF-positive cells that are also PAX3-positive averaged across all samples. E) Double immunofluorescent staining shows PAX3 and MITF co-expressing melanocytes (yellow-orange) in the bulb of the hair follicle of normal skin. Note the single MITF-labelled melanocytes (green) at the base of the hair. doi:10.1371/journal.pone.0009977.g002 phenotype respectively. Upon analysis of the distribution of double-labelled cells we found that HES1 was expressed in a proportion of PAX3-positive melanocytes, while cells that were only HES1-positive were identified as keratinocytes [31,32] (Figure 3A). The number of PAX3-positive melanocytes co- expressing HES1 was lowest in the matrix of the hair bulb (10.5%), PLoS ONE | www.plosone.org 4 April 2010 | Volume 5 | Issue 4 | e9977