Journal: Nature Communications
Article Title: Molecular profiling of driver events in metastatic uveal melanoma
Figure Lengend Snippet: Copy number analysis. a Copy number profiles of each tumor. Differences in color intensity depend on copy number amplitude and tumor purity. b Broad copy number changes enriched in the metastases ( n = 32) compared with TCGA tumors ( n = 80). Two-tailed Fisher’s exact tests with adjustment for multiple testing using the Benjamini–Hochberg method. c Two primary tumors compared with matched metastases. d Focal deletions of CDKN2A in two samples. e RNA-seq from the UM9 metastasis and corresponding PDX showing the region with focal CDKN2A deletion. f Genes in recurrent arm-level copy number aberrations ranked by associations between gene expression and copy number that were consistent in this cohort and TCGA tumors, and further ranked by protein–protein interaction network degree from the Human Protein Reference Database (HPRD), and additionally by presence of any associations with worse survival. The top three candidates are shown in each region. Connecting lines represent protein interactions of the highest ranked gene per region. Blue represents regions of loss and red regions of gain. Summarized representations of copy number profiles per region show the relative numbers of gain and loss events, with the inner circle representing TCGA samples and the outer our cohort. For the metastatic cohort, n = 28 samples with matching DNA and RNA data were included. g Pathways enriched among the combined set of the top 10 genes per region of gain or loss. h Functional interrogation by siRNA of main candidate genes whose expression is elevated due to copy number alteration. Cells were counted or viability was measured at 72 h, 96 h and 96 h for the cell lines UM22, MP-41, and 92-1, respectively, after transfection of the siRNA pools. n = 3 samples were transfected independently, for each cell line. Data are presented as mean values ± standard error of the mean (SEM). Two-way ANOVA was used to estimate differences, taking into account both cell line and target gene as variables. q values were calculated using Benjamini–Hochberg correction, taking into account all genes in h as well as those in Supplementary Fig. 3c , which shows other candidates of interest investigated. Dotted lines indicate q = 0.05.
Article Snippet: The siRNA duplexes were purchased from Dharmacon (Thermo Fisher Scientific, Waltham, MA, USA) and the lipid based transfection was performed with Lipofectamine-RNAiMAX (Thermo Fisher Scientific, Waltham, MA, USA) using 1 pmol of siRNA per well of a 96-well plate as per the guidelines provided by manufacturer.
Techniques: Two Tailed Test, RNA Sequencing Assay, Expressing, Functional Assay, Transfection