JCS013912 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Comparative melanocyte quantification and characteristic of wild-type and Nf1^+/− mice and primary cultures. (A) Quantitative comparison of Kit⁺CD45⁻ populations in wild-type and Nf1^+/− primary cultures. Cells obtained from the neonatal dermal suspensions of C57BL6-Nf1^+/−× C57BL10 progeny were cultured for 10 days in the presence of TPA, bFGF and dbcAMP, which were withdrawn 72 hours prior to FACS analysis. Cells were double-labeled with APC-CD45 and PE-CD117 (Kit) antibody as described and the Kit⁺CD45⁻ population analyzed for pooled wild-type and Nf1^+/− cells. Results are representative of multiple independent measurements. (B) Comparison of melanocyte numbers between wild-type and Nf1^+/− murine skin. C57Bl6-Nf1^+/− mice were intercrossed with TgDct-LacZ mice. Cryosections from TgDct-LacZ and Nf1^+/−; TgDct-lacZ progeny were stained for β-gal activity and visualized under bright field microscopy. The number of melanocytes in ten longitudinally-sectioned hair follicles of a wild-type and Nf1^+/− mouse were counted and averaged. Error bars represent standard deviation from the mean. (C) Erk expression and activation in Mitf^Mi-wh/Mitf^Mi-wh mixed primary cultures. Mixed primary cultures from dermal suspensions of Mitf^mi-wh/Mitf^mi-wh neonates were cultured for 10 days in the presence of TPA, bFGF and dbcAMP, which were withdrawn 72 hours prior to stimulation and cell lysis. Mitf^Mi-wh/Mitf^Mi-wh primary cultures were either not stimulated (US) or stimulated with SCF/Kit ligand for 5, 15 and 30 minutes prior to cell lysate preparation. 5 µg of total protein was loaded onto and SDS-PAGE gel and western blotting used to detect phosphorylated Erk and Erk2 expression. Primary cultures from wild-type (+/+) and Nf1^+/− (+/−) cells were used as positive controls. (D) Expression of neurofibromin in Nf1^+/− cells. 5 µg of cell lysate from 10-day-old wild-type (+/+) and Nf1^+/− (+/+) primary cultures were probed for neurofibromin protein expression by western blotting. (E) SCF/Kit ligand concentrations in wild-type and Nf1^+/− cell culture medium. Conditioned culture medium from wild-type (+/+) and Nf1^+/− (+/−) cells cultured for 8 days was used for determining concentrations of murine SCF/Kit ligand by ELISA.

• Supplemental Figure S2 -

  Fig. S2. Comparison of Tyrp1 expression in isolated single +/+ and Nf1^+/− melanocytes. Comparison of Tyrp1 expression between three individual wild-type melanocytes (top row) and three individual Nf1^+/− melanocytes (bottom row) under identical experimental and photomicroscopic conditions. Increased Tyrp1 expression is suggested in the middle and right Nf1^+/− melanocytes but heterogeneity of expression makes this difficult to conclude.