Journal: The Journal of Clinical Investigation

Article Title: Comparative oncogenomics identifies tyrosine kinase FES as a tumor suppressor in melanoma

doi: 10.1172/JCI91291

Figure Lengend Snippet: (A) Analysis of FES expression in 474 melanoma clinical samples from the TCGA cohort. The left panel shows FES mRNA levels ordered from the highest (red) to the lowest (green). The middle panel shows DNA methylation profile obtained from 19 array probes located in the CpG sites of FES. The schematic above shows a representation of the FES locus, with UTR regions in white and exons in black. CpG positions are shown as red stripes. The right panel depicts the copy number status of the FES locus split into cases that show loss (in blue) and gain (in red) and samples in which the CNA status of FES was not assessed (in gray). (B) FES expression in short-term melanoma cultures (MM) and 3 normal melanocyte cultures (NM). Upper graph shows expression of FES mRNA levels as determined by RT-qPCR. Values are normalized to the mean RNA level of normal melanocytes, which was set to 1. Error bars show mean ± SD (n = 2). The middle panel shows Western blot analysis of FES. Actin served as a loading control. The bottom panel shows methylation profile of short-term melanoma cultures and normal melanocytes as determined by bisulfite sequencing of 20 CpG sites located at positions ranging from –72 to +115 from the FES′ TSS. BRAFV600E (B) and NRASQ61K,L,R (N) mutational status is indicated on top of the sample name. (C) Expression of FES in MM031 cell culture after treatment with demethylating agent decitabine or its vehicle. The upper panel shows FES mRNA levels assessed by qRT-PCR. Western blot analysis in the 2 lower panels shows FES protein levels. GAPDH served as a loading control.

Article Snippet: For bisulfite conversion, we used the EZ DNA Methylation Gold Kit (Proteigene) according to the manufacturer’s instructions.

Techniques: Expressing, DNA Methylation Assay, Quantitative RT-PCR, Western Blot, Methylation, Methylation Sequencing, Cell Culture

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Beneficial Effects of Myo -Inositol Oxygenase Deficiency in Cisplatin-Induced AKI

doi: 10.1681/ASN.2016070744

Figure Lengend Snippet: Cisplatin treatment per se upregulates MIOX via demethylation of its promoter. WT mice treated with cisplatin had an increased renal expression of MIOX, and it was confined to the tubules (A–C). No significant MIOX expression was seen in glomeruli. Genomic DNA was isolated from kidneys of WT and cisplatin-treated mice and subjected to bisulfite treatment. Promoter fragments were isolated by PCR, cloned into pGEM-T plasmid, and transformed in DH5α cells. Ten clones from each variable were selected and subjected to nucleotide sequencing to assess the efficiency of conversion (cytosine to thymine) of CpG dinucleotides. Nucleotide analyses revealed eight CpG dinucleotides at −19, −22, −42, −48, −73, −424, −436 and −456 bp sites. In WT, untreated control mice samples, CpG at −22 and −436 bp were found to be unmethylated, whereas six others were methylated (D and E). The samples from cisplatin-treated mice were found to have all of the CpG dinucleotides unmethylated, except for −424 and −456. Overall, the efficiency of conversion of C to T nucleotide was >95%. The percentages of methylation of individual dinucleotide residues are presented as bar graphs, which indicate that cisplatin induces hypomethylation that is likely responsible for the MIOX upregulation (F).

Article Snippet: Sigma-Aldrich: cisplatin (# P4394), anti– β -actin antibody (# A5441), Caspase-3 assay kit, Colorimetric (# CASP-3C), poly-(deoxyinosinic-deoxycytidylic) acid sodium salt (# P4929), NAC (# A7250); BioAssay Systems: QuantiChrom creatinine assay kit (# DICT-500) and QuantiChrom urea assay kit (# DIUR-500); Life Technologies: Power SYBR Green PCR Master Mix (# 4367659), Chloromethyl derivative of 2′, 7′-dichlorofluorescein diacetate (CM-H 2 DCF-DA, # C6827), 4′-6-diamidino-2-phenylindole (DAPI, # D1306), and TRIzol Reagent (# 15596026); Cell Signaling Technology: anti–cleaved caspase-3 antibody (# D175), anti-p53 (1C12) antibody (# 2524), and anti-p65 antibody (# D14E12); Abcam: anti–lamin B1(ab16048); Enzo Life Sciences: DNA methylation gold kit (# D5005); Santa Cruz Biotechnology: anti-Bax antibody (# SC-526), anti–NOX-4 antibody (# SC-21860), anti-p53 antibody (# SC-6243); Roche Diagnostic: In Situ Cell Death Detection Kit, Fluorescein (# 11684795910); Cayman Chemicals: GSH assay kit (# 703002); Promega Corporation: NF- κ B EMSA oligo (# E329A) and pGEM-T plasmid vector; Perkin Elmer: ATP γ -32p (# BLU002A); and Thermo Scientific: polynucleotide kinase (EK0032).

Techniques: Expressing, Isolation, Clone Assay, Plasmid Preparation, Transformation Assay, Sequencing, Methylation