Fig. 6. Effects of reduction of neurofibromin in immortalized murine melanocytes. (A) siRNA-mediated depletion of neurofibromin in melan-a cells. Melan-a cells at a cell density of 2 x10⁵ cells per well in a six-well plate were transfected with three different siRNAs (lanes 1-3) targeting Nf1. Cells were also transfected with a control, scrambled siRNA (lane M) or activated with 40 µM TPA (lane U) for 15 minutes prior to lysis. Cells were lysed 72 hours post-transfection and evaluated for siRNA-mediated knockdown of neurofibromin with western blotting. (B) Melanogenic gene expression in melan-a cells treated with neurofibromin siRNA. Melan-a cells at a cell density of 2 x10⁵ cells per well in a six-well plate were transfected with the same siRNAs targeting Nf1. RNA isolated from cells 48 hours post-transfection was evaluated for Tyr, Tyrp1 and Dct expression by real time RT-PCR. Experiments were performed in triplicate, with error bars representing standard deviation from the mean. The fold increase in gene expression for each siRNA gene pair is relative to the expression level measured for the same gene in cells transfected with the control, scrambled siRNA (Control) described above.