Journal: Journal of the American Society of Nephrology : JASN
Article Title: Beneficial Effects of Myo-Inositol Oxygenase Deficiency in Cisplatin-Induced AKI
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 DH5 α 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-H2 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: Mouse Assay, Expressing, Isolation, Polymerase Chain Reaction, Clone Assay, Plasmid Preparation, Transformation Assay, Sequencing, Methylation