Journal: bioRxiv

Article Title: The shh limb enhancer is activated in patterned limb regeneration but not in hypomorphic limb regeneration in Xenopus laevis

doi: 10.1101/2022.12.27.522067

Figure Lengend Snippet: (A) Upper panel: Map of MFCS1-luc2 used for assays in (B). Lower panel: Alignment of MFCS1 highly conserved sequences. Nucleotides identical in more than two sequences are shaded in grey. Conserved Hox-binding motifs are boxed. A light bule line underlies the 14-thymidine repeat in X. tropicalis (See Materials and Methods for details.). (B) Firefly luciferase activity of MFCS1-luc2 using a minimum promoter (minP) or shh promoter (shh pro) was normalized to Renilla luciferase control activity. The mean value of mock samples by the control construct with the minimum promoter was adjusted to 1.0. Error bars, standard deviation from the mean (n=3). *Significant increase from mock (p<0.01). **Significant increase in Fgf2+Hoxd13, Fgf4+Hoxd13 and Fgf8+Hoxd13 from sole Fgf2, Fgf4 and Fgf8, respectively (p<0.01). (C) Methylation pattern of endogenous and transgene MFCS1 (line 1). Samples were obtained from the autopod (pink). Each circle corresponds to a CpG site (see ). Lower panel: Increases in average methylation rate of the MFCS1 sequences after metamorphosis. (D) Model for epigenetic silencing of gene expression through DNA methylation of MFCS1. Hoxd13 activates hypomethylated MFCS1 and results in transcription of a gene under control of MFCS1 in the tadpole limb (left). However, Hoxd13 is unable to activate highly methylated MFCS1 in a froglet limb and results in inactive transcription of the gene (right).

Article Snippet: The genomic DNA was treated with sodium bisulfite using an EZ DNA Methylation-Gold Kit following the protocol of the manufacturer (Zymo Research).

Techniques: Binding Assay, Luciferase, Activity Assay, Construct, Standard Deviation, Methylation, Expressing, DNA Methylation Assay

Journal: bioRxiv

Article Title: The shh limb enhancer is activated in patterned limb regeneration but not in hypomorphic limb regeneration in Xenopus laevis

doi: 10.1101/2022.12.27.522067

Figure Lengend Snippet: (A) Methylation-sensitive restriction digestion visualized by agarose gel electrophoresis. Plasmid DNA of the MFCS1- shh promoter- luc2 reporter was treated with CpG methyltransferase (M. SssI, NEB) according to the manufacturer’s protocol. Methylation efficiency of the plasmid DNA was evaluated by digestion with methylation-sensitive (HpaII) and -insensitive (MspI) restriction enzymes. The CpG methylation prohibited the digestion of the plasmid DNA specifically with HpaII. M, l/HindIII marker. (B) Luciferase activity in NIH3T3 cells transfected with the methylated or non-treated MFCS1-luc2 reporter constructs and the Hoxd13 expression construct. The mean value of luciferase activity in cells transfected with the non-treated reporter and mock expression constructs was set to 1.0. Error bars: standard deviation from the mean (n=3). While a significant increase of Hoxd13 -induced luciferase activity was observed in cells transfected with the unmethylated reporter construct (*p<0.01), being consistent with , there was no significant increase in cells transfected with the methylated reporter construct despite Hoxd13 expression. N. S., not significant.

Article Snippet: The genomic DNA was treated with sodium bisulfite using an EZ DNA Methylation-Gold Kit following the protocol of the manufacturer (Zymo Research).

Techniques: Methylation, Agarose Gel Electrophoresis, Plasmid Preparation, CpG Methylation Assay, Marker, Luciferase, Activity Assay, Transfection, Construct, Expressing, Standard Deviation

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