Figure 4

Long-term N-RAS^61K expression diminishes melanocyte marker expression and increases neuronal gene expression and pluripotency features. (a) Heatplot displaying the RNA expression of melanocyte differentiation markers in N-RAS^61K cells stimulated with doxycycline for 6, 14 and 28 days and in N-RAS^61K-AR cells. (b) Confirmation of differential gene expression by real-time PCR using primers directed against Tyrp1, Mlana, Dct, Sox10 and Mitf. (c) Heatplot displaying the RNA expression of neuronal genes in the same samples as described in (a). (d) Real-time analysis showing differential expression of Nefl and Smarca. For (a and c), the values are color coded using a green–red scale, where green indicates low expression and red indicates high expression. (e) Real-time PCR analysis of Pdpn expression (upper panel) and RT-PCR analysis of Nanog expression (40 cycles, lower panel) in N-RAS^61K cells stimulated with doxycycline for 6, 14 and 28 days and in N-RAS^61K-AR cells. Hprt served as a control. (f) Nanog-driven GFP expression in N-RAS^61K and N-RAS^61K-AR cells, transiently transfected with the PL-SIN-EOS-S(4+)-eGFP vector. Scale bars, 100 μm. (g) Determination of the nuclear/cytoplasmic ratio of N-RAS^61K and N-RAS^61K–AR cells. Left: Confocal images of N-RAS^61K and N-RAS^61K-AR cells. Arrows, 50 μm. Right: Averaged cellular, nuclear or cytoplamic volume of N-RAS^61K and N-RAS^61K-AR cells upon generation of confocal stacks (see Supplementary Movie S3). Compartment volumes were determined using Volocity v4 software, P=0.0076 (cell), P=0.0052 (cytoplasm); n=6. *P<0.05; **P<0.01, ***P<0.001