Fig 2: Immunohistochemistry results for Nodal staining. (A) Representative results show immunohistochemistry staining (brown) for Nodal in dysplastic nevi and LCMN from pediatric patients (20× original magnification); (B) Box plot representation shows that median staining scores for Nodal were similar between the two different groups analyzed.

Fig 3: HeMn melanocyte derived melanosomes (MDM) affect Nodal expression and signaling components in C8161. (A) Microphotograph (40× original magnification) shows MDM containing globules (red arrows) produced by HeMn melanocytes; (B) C8161 melanoma cells treated with different concentrations of MDM collected from HeMn melanocytes showed reduction in Nodal expression compared to untreated control C8161 cells. To mimic the anti-oxidant effect of free radical scavengers contained in MDM, C8161 cells were treated with N-acetyl-l-cysteine (NAC) known to generate the anti-oxidant, glutathione; (C) Western Blot analysis show that treatment of C8161 melanoma cells with 5 mM NAC resulted in reduced levels of Nodal, P-SMAD and P-ERK1/2 compared to untreated control C8161 cells; (D) Treatment of C8161 cells with synthetic melanin, also known to have anti-oxidant properties, resulted in a dose-dependent reduction of Nodal, P-SMAD and P-ERK1/2 levels compared to untreated control C8161 cells. Numbers above WB bands represent densitometric units, normalized to actin loading control, total SMAD2/3 or total ERK1/2, as appropriate, relative to control.

Fig 4: Effect of treatment of C8161 melanoma cells with HeMn melanocyte conditioned medium (HeMn-CM). (A) C8161 cells treated with HeMn-CM show morphologic changes such as dendritic shape with long cytoplasmic extensions reminiscent of melanocyte differentiation. (40× original magnification); (B) Although, Melanoma Differentiation Associated protein 7 (MDA-7) was detected in C8161 cells treated with HeMn-CM, the melanin synthesis associated enzyme tyrosinase was not; (C) C8161 cells treated with HeMn-CM showed similar proliferation rate and cell viability as non-treated control C8161 cells making potential toxic effect of HeMn-CM on C8161 unlikely; (D) Western blot analysis show that HeMn-CM treated C8161 express lower levels of Nodal, P-SMAD2 and P-ERK1/2 compared to untreated control C8161 cells. Numbers below WB bands represent densitometric units, normalized to actin loading control, total SMAD2/3 or total ERK1/2, as appropriate, relative to control.