SUPPLEMENTARY INFORMATION

Supplementary Figure S1 (jpeg 467K)

TYRP-1 immunohistochemical expression. A. Foreskin epiderm showing strong and specific TYRP-1 positivity of normal melanocytic cells. B, C, D. NCG22, 26 and 21 showing absence or very weak TYRP-1 expression of the intradermal and/or junctional naevic cell nests contrasting with strong positivity of normal epidermal melanocytes.

Supplementary Tables S1a–e (xls 42,602K)

Table S1a. Microarray data from "in-house made" slides. Analyses have been performed in triplicate for each CMN sample and each value is expressed as a mean of the normalized log2-ratios of CMN triplicate with its respective Standard Deviation (SD). "Invalid" refers to data not taken into account because of a signal/background ratio less than 1,5 or artefact-associated spots eliminated by both visual and software guided flags. NRAS mutated sample names are in grey, BRAF mutated samples in red, BRAF translocated samples in green, NCG10 in orange and NCG25 in blue.

Table S1b. Selection of genes showing a common dysregulation in at least 21 out of the 27 CMN analyzed. The "SAM one class" method with the default parameters was used in order to identify genes differentially expressed among all the samples hybridized on "in-house made" slides. A second more stringent selection was then performed from this list by using a cut-off of absolute normalized fold change >1.5 (log2 normalized ratios >0.6) in any direction in 75% (21/27) of the samples. Each CMN was hybridized in triplicate, and the reported values for each CMN are expressed as a mean of the normalized log2-ratios of CMN triplicate with its respective SD. NRAS mutated sample names are in grey, BRAF mutated samples in red, BRAF translocated samples in green, NCG10 in orange and NCG25 in blue.

Table S1c. Selection of genes showing a common dysregulation in at least 21 out of the 27 CMN analyzed, but after having suppressed all "not identified" and redundant spots. Same data as the ones given in Table S1b, but after having suppressed all "not identified" and redundant spots.

Table S1d. Subset of genes differentially expressed between the two classes of unpaired samples (6 BRAF+ samples and NCG25 versus the remaining samples including NCG10) identified by Significance Analysis of Microarray ("SAM two unpaired classes"). A subset of genes differentially expressed between the two classes of unpaired samples (the 6 BRAF+ samples and NCG25 versus the remaining samples including NCG10) was identified by using SAM method (which ensures that the number of false discoveries will not exceed 0.01%). Each CMN was hybridized in triplicate, and the reported values for each CMN are expressed as a mean of the normalized log2-ratios of CMN triplicate with its respective SD. NRAS mutated sample names are in grey, BRAF mutated samples in red, BRAF translocated samples in green, NCG10 in orange and NCG25 in blue.

Table S1e. Subset of genes differentially expressed among the two classes of unpaired samples (6 BRAF+ samples and NCG25 versus the remaining samples including NCG10) identified by Significance Analysis of Micro-array("SAM two unpaired classes"), but after having suppressed all "not identified" and redundant spots. Same data as the ones given in Table S1d, but after having suppressed all "not identified" and redundant spots.

Supplementary Tables S2a and b (xls 8,583K)

Table S2a. Microarray data from "HEEBO" slides of the 4 BRAF mutated samples. Analyses have been performed with dye swap for each CMN sample and the reported values for each CMN are expressed as a mean of the normalized log2-ratios of CMN duplicate with its respective SD. "Invalid" refers to data not taken into account because of a signal/background ratio less than 1,5 or artefact-associated spots eliminated by both visual and software guided flags.

Table S2b. Selection of genes showing a common dysregulation in at least 3 out of the 4 BRAFV600E+ CMN analyzed. The "SAM one class" method with the default parameters was used in order to identify genes differentially expressed among the 4 BRAF mutated samples hybridized on "HEEBO" slides. A second selection was then performed from this list by using a cut-off of absolute normalized fold change >1.5 (log2 normalized ratios >0.6) in any direction in 75% ( 3/4) of the samples. Analyses have been performed with dye swap for each CMN sample and the reported values for each CMN are expressed as a mean of the normalized log2-ratios of CMN duplicate with its respective SD. Results are reported for 782 spots corresponding to 560 genes.

Supplementary Table S3 (doc 131K)

List of gene up- and down-regulations leading to either activation or inhibition of the most significant cellular processes. This profile was obtained from BRAF V600E+ CMN cases and with use of "HEEBO" slides.

Ratio: mean of dye-swapped ratios of the 4 BRAF V600E+ CMN. Up-regulated genes: gene up-regulation leading to either activation or inhibition of the cited biological processes. Down- regulated genes: gene down-regulation leading to either activation or inhibition of the cited biological processes.

Supplementary Table S4 (doc 50K)

Comparison between microarray and qRT-PCR data obtained for the 4 BRAF mutated CMN cases and regarding 8 significantly dysregulated genes. The mean and standard deviation (SD) values given in the "HEEBO" column were obtained from all expression ratios (dye-swaps as well as redundant signals for each reported gene) for each of the 4 BRAF mutated CMN cases. qRT-PCR values represent the mean of the ratios of the normalised expression for each tested transcript in CMN triplicate samples to that in "normal pool", and their respective SD.

In 7 out of 8 tested genes (ARL4C, ETS1, PMP22, TACC1, TFRC, TIMP3 and GAPDH), statistic analysis of qRT-PCR results confirmed the significant gene dysregulations obtained by microarray analysis in all samples. For the remaining gene (CCND1), statistical significance was not reached in the 4 samples. In our series, we obtain a global concordance of >90%, comparable to other studies (Pavey et al, 2004).

Supplementary Materials and Methods (doc 72K)

Supplementary Materials and Methods for cell culture ; list of forward and reverse primers and respective PCR conditions used to perform PCR investigations and sequencing, as well as quantitative Real-Time PCR ; supplementary Materials and Methods for microarrays experiments and supplementary Materials and Methods for immunohistochemical studies.