melanocyte cell basal medium  (PromoCell)

Journal: Nature Communications

Article Title: Massively parallel reporter assays of melanoma risk variants identify MX2 as a gene promoting melanoma

doi: 10.1038/s41467-020-16590-1

Figure Lengend Snippet: a Variants that were tested in MPRA from the Chr21q22.3 melanoma locus are shown relative to the genomic position of MX2 . Only the 19 variants located in the first intron of MX2 coming from the primary GWAS signal are shown (the other three from a secondary signal are located upstream of the MX2 genic region). ChromHMM annotation (Primary Core Marks segmentation) of Penis Foreskin Melanocyte Primary Cells from Roadmap Epigenomics Project is shown (Red/OrangeRed: Active_TSS/Flanking_Active_TSS, Yellow/GreenYellow: Enhancers/Genic_enhancers, Green/DarkGreen: Strong_transcription/Weak_transcription). Melanoma FAIRE-seq track of 11 samples is from a study by Verfaillie and colleagues . Ensembl predicted transcripts from archive 75 are shown. b Transcriptional activity of 145 bp sequences encompassing rs398206 from MPRA are shown as normalized tag counts (log 2 (RNA TPM/DNA TPM)). Results from UACC903 melanoma cells are shown for both alleles in forward (For) and reverse (Rev) directions, where results from promoter and enhancer constructs were combined. Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots. Density is reflected in the width of the shape. c Individual luciferase activity assays of 145 bp sequences encompassing rs398206 is shown for UACC903. pGL4.23 construct including minimal TATA promoter was used. One representative set is shown from three biological replicates. Mean with SEM, n = 6. All constructs are significantly higher than pGL4.23 (TATA) control ( P < 0.0001, two-tailed, unpaired t-test assuming unequal variance). d eQTL plot of MX2 levels in primary melanocytes in relation to rs398206 is shown for three genotype groups. Center lines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. P -value and slope were derived from linear regression with no multiple-testing correction applied. Source data are provided as a Source Data file.

Article Snippet: Melanocyte cells were grown in Dermal Cell Basal Medium (ATCC PCS-200-030) supplemented with Melanocyte Growth Kit (ATCC PCS-200-041) and 1% amphotericin B/penicillin/streptomycin (120-096-711, Quality Biological) as described before .

Techniques: Activity Assay, Construct, Software, Luciferase, Control, Two Tailed Test, Derivative Assay

Journal: Nature Communications

Article Title: Massively parallel reporter assays of melanoma risk variants identify MX2 as a gene promoting melanoma

doi: 10.1038/s41467-020-16590-1

Figure Lengend Snippet: a Ingenuity Pathway Analysis of differentially expressed genes from MX2 -high vs. MX2 -low melanocytes from 106 individuals. 252 differentially expressed genes (FDR < 1% and >2-fold change) between MX2 -high and MX2 -low melanocytes (top and bottom quantile based on MX2 levels; n = 28 each) from 106 individuals were used as input for the analysis. Enrichment P -values are based on a two-sided Fisher’s exact test with no multiple-testing correction applied. (B-C) Cell growth and movement of human primary melanocytes C23 ( b ) or melanoma cell line UACC2545 ( c ) infected with an inducible lentiviral construct of MX2 cDNA or Empty pINDUCER20 vector were measured on xCELLigence system. Cell Index values were normalized relative to those at the time of doxycycline addition (dotted vertical line: Dox). The amount of doxycycline (dox) is shown in ng/ml and color-coded. Mean Normalized Cell Index (colored dots) and SD (gray vertical lines) are plotted ( n = 3). A representative set of three biological replicates is shown. d Ingenuity Pathway Analysis of differentially expressed genes from RNA sequencing of MX2 overexpressed vs. control melanocytes from 3 individuals. 158 differentially expressed genes (FDR < 10%) between MX2 -overexpressing (100 ng/ml doxycycline) vs. control (no doxycycline) melanocytes using 3 biological replicates for 3 individuals were used as input for the analysis. Significantly enriched canonical pathways (P < 0.05 and |Z-score| >1) are color-coded for the direction of effect relative to MX2 -high melanocytes ( a ) or MX2 -overexpressing melanocytes ( d ). Enrichment P-values are based on a two-sided Fisher’s exact test with no multiple-testing correction applied. A weaker to stronger shade of each color represent the relative magnitude of Z -scores: Positive Z -score between 1 and 2.646 and negative Z -score between −1 and −3.464 ( a ) or positive between 1 and 1.134 and negative between −1.342 and −2.236 ( d ), where lightest red is closer to 1 and lightest blue is closer to −1. ( e ) Representative pictures of adult fish from GFP or MX2 group. Pictures were taken at week 10 post-injection. f Melanoma-free survival curves of a zebrafish melanoma model (Tg( mitfa:BRAF V600E ), p53 −/−, mitfa −/−). The fish were injected at the one cell stage with either miniCoopR mitfa:MX2 or miniCoopR mitfa:EGFP and monitored weekly for melanoma formation. The percentage of melanoma-free fish was combined from three independent experiments and plotted. Log-rank test was used. Source data are provided as a Source Data file.

Article Snippet: Melanocyte cells were grown in Dermal Cell Basal Medium (ATCC PCS-200-030) supplemented with Melanocyte Growth Kit (ATCC PCS-200-041) and 1% amphotericin B/penicillin/streptomycin (120-096-711, Quality Biological) as described before .

Techniques: Infection, Construct, Plasmid Preparation, RNA Sequencing, Control, Injection