Legends to Supplementary Figures and Tables.
Supplementary Figure 1. Gene expression in 501Mel and 1205Lu cells. A. Heatmap illustrating expression of 31 genes in 96 cells from 501Mel monolayers. The colour key showing the log2 expression values is shown right of the panel.B. Heatmap illustrating expression of 59 genes in 95 cells from 1205Lu monolayers. The colour key showing the log2 expression values is shown right of the panel.
Supplementary Figure 2. Reproducibility of Single cell analyses.A-B.Correlation of gene expression values for the same set of cDNAs analysed for the indicated genes either within a single Biomark qPCR plate (A) or on separate Biomark qPCR plates (B). The Pearson correlation coefficients are indicated on each panel. C-D.Identification of target genes directly regulated by MITF. C.Enlargement of cluster C of Fig. 3B showing genes strongly correlated with MITF in 501Mel derived tumours. D. UCSC gene browser views of 3HA-MITF ChIP-seq for the indicated gene loci. Arrows indicate representative MITF occupied sites. For sake of space the corresponding control input and 3HA-ChIP tracks have been omitted. The data were generated as described in 1
Supplementary Figure 3.Gene expression in cells from1205Lu-derivedmelanospheres. A. Heatmap illustrating expression of 81 genes in 95 cells from 1205Lu-derived spheres. The asterisks *** indicate a group of cells showing higher expression of many of the tested transcripts. B. Identification of co-regulated genes in 1205Lu cells grown as melanospheres Heatmap illustrating correlated changes in gene expression in single cells from 1205-derived spheres. The median expression of each gene relative to quartiles of expression is shown above the heatmap. The colour key showing the Pearson correlation coefficient is shown to the right of the figure.
Supplementary Figure 4. Gene expression in cells from1205Lu-derived tumours.A.The left hand panels show images of tumours generated from 501Mel or 1205Lu cells as indicated after excision from the animals. The right hand panel show images from hematoxyline-eosine stained sections through the tumours. B. Heatmap illustrating expression of 114 genes in 95 cells from 1205-derived tumours. Asterisks *** indicate cells expressing higher levels of many of the tested transcripts.
Supplementary Figure 5.Comparative gene expression in cells from1205Lu and 501Mel-derived tumours. Heatmap illustrating comparative expression of 113 genes from 92 cells from 501Mel and 95 cells from 1205Lu-derived tumours. Cluster A: Proliferative signature and MITF target genes showing preferential expression 501Mel cells. Cluster B: Sub-group of invasive signature genes showing preferential expression 1205Lu cells. Cluster C: Second sub-group of invasive signature genes showing preferential expression 1205Lu cells. Asterisk * indicates a single 501Mel cell expressing invasive signature genes. Asterisk * indicates a single 501Mel cell expressing invasive signature genes.
Supplementary Figure 6.Comparative gene expression in cells from 1205Lu and 501Mel-derived melanospheres and tumours. A. Heatmap illustrating comparative expression of 76 genes in 180cells from 501Mel-derived melanospheres (MS, 88 cells) and tumours (TUM, 92 cells). Genes showing preferential or depleted expression in either of the two conditions are indicated. B.Heatmap illustrating comparative expression values of 77 genesin 190cells from 1205Lu-derived melanospheres (MS, 95 cells) and tumours (TUM, 95 cells). Genes showing preferential or depleted expression in either of the two conditions are indicated.
Supplementary Figure 7. Changes in gene expression under different culture conditions seen in single cells are observed at the population level. A-B. Ratios of expression of the indicated genes measured by qPCR in 501Mel cells between monolayers and tumours, monolayers and melanospheres and melanospheres and tumours using independent RNA populations. C-D. Ratios of expression of the indicated genes measured by qPCR in 1205 cells as described in panel A. All values represent results from three independent qPCR reactions with less than one cycle of difference in the Ct value.
Supplementary Table 1. List of significantly differentially expressed genes in 1205Lu compared to 501Mel. Genes preferentially expressed in 1205Lu are shown on page 1 and genes preferentially expressed in 501Mel on page 2. Page 3, list of genes whose expression was assayed in this study. The list of genes is shown along with indications of whether they belong to the Widmer profile, or whether they are preferentially expressed in 501Melor 1205Lu. Primer sequences designed by the authors are indicated. When not indicated, the primers were designed and provided by the Fluidigm corporation. Also indicated on the table are whether the genes are direct MITF targets based on the presence of a MITF occupied site in ChIP-seq experiments within +/- 20kb of the transcription start site in 501Mel cells. Lastly, the table indicates whether the genes were de-regulated by si or shMITF silencing in 501Mel cells. The siMITF data is as presented in 1and the shMITF data is in preparation.
1Strub T, Giuliano S, Ye T, Bonet C, Keime C, Kobi D et al. Essential role of microphthalmia transcription factor for DNA replication, mitosis and genomic stability in melanoma. Oncogene 2011; 30: 2319-2332.