Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Dynamic DNA methylation of matrix metalloproteinase-9 in the development of diabetic retinopathy

doi: 10.1038/labinvest.2016.78

Figure Lengend Snippet: High D-glucose hypomethylates MMP-9 promoter in retinal endothelial cells, and increases Dnmt1 binding at the same region of the promoter. The levels of 5mC were quantified in retinal endothelial cells by methylated DNA immunoprecipitation kit from Epigentek, and primers for (a) promoter region (−720 to −547), and (b) intragenic CpG island region (1487 to 1701) of MMP-9 gene. The total genomic DNA from cells in 5mM glucose served as an input DNA control. (c) Dnmt1 binding at the MMP-9 promoter was quantified using Dnmt1 monoclonal antibody, followed by amplification of the promoter region by q-PCR. IgG was used as a negative antibody control (indicated as ^), and Ct values were normalized with the values from input by ddCt method. Product size was confirmed on an agarose gel. Values are represented as mean ± SD from 4–5 samples in each group. 5mM and 20mM= 5mM or 20mM D-glucose, Mann= 20mM mannitol. *p< 0.05 compared to 5mM D-glucose.

Article Snippet: Sonicated DNA was immunoprecipitated for 5mC/5hmC using methylated/ hydroxymethylated DNA Immunoprecipitation (MeDIP/hMeDIP) kits (EPIGENTEK, Farmingdale, NY).

Techniques: Binding Assay, Methylation, Immunoprecipitation, Control, Amplification, Agarose Gel Electrophoresis

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Dynamic DNA methylation of matrix metalloproteinase-9 in the development of diabetic retinopathy

doi: 10.1038/labinvest.2016.78

Figure Lengend Snippet: High D-glucose increases hydroxymethylation of MMP-9 promoter and Tet activity. (a) The levels of 5hmC were quantified in retinal endothelial cells using hMeDIP immunoprecipitation kit. (b) The enzyme activity of Tet was determined in 10–20μg sample using TET Activity/Inhibition Assay Kit, and the values obtained from cells in 5mM glucose are considered as 100%. (c) MMP-9 mRNA was quantified by q-PCR. Values are represented as mean ± SD from 4–5 cell preparations, with each measurement made in duplicate. 5mM and 20mM= 5mM or 20mM D-glucose; 5+/2-HG and 20+/2-HG= cells incubated in 5mM or 20mM D-glucose in the presence of 500μM 2-HG; Mann= 20mM mannitol; L-Gl=cells in 20mM L-glucose. *p< 0.05 compared to 5mM D-glucose

Article Snippet: Sonicated DNA was immunoprecipitated for 5mC/5hmC using methylated/ hydroxymethylated DNA Immunoprecipitation (MeDIP/hMeDIP) kits (EPIGENTEK, Farmingdale, NY).

Techniques: Activity Assay, Immunoprecipitation, Inhibition, Incubation

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Dynamic DNA methylation of matrix metalloproteinase-9 in the development of diabetic retinopathy

doi: 10.1038/labinvest.2016.78

Figure Lengend Snippet: Diabetes hypomethylates MMP-9 promoter, but the binding of Dnmt1 in the same region is increased. (a) The levels of 5mC were quantified in the promoter (−640 to −356) and the intragenic CpG island (1,361 to 1,600) regions of the MMP-9 gene by methylated DNA immunoprecipitation kit. Total genomic DNA obtained from normal mouse retina served as input DNA control. Gene transcripts of (b) MMP-9 , and (c) Dnmts were quantified by SYBR green based q-PCR using β-actin as the housekeeping gene. (d) The binding of Dnmt1 at the MMP-9 promoter region was measured in the crosslinked retina by immunoprecipitating the samples with Dnmt1 antibody, followed by amplification of the promoter region by q-PCR. IgG was used as a negative antibody control (indicated as ^), and Ct values were normalized with the values from input by ddCt method. Values are represented as mean ± SD from 5–7 mice in each group. Norm and Diab= retina from nondiabetic normal mouse or from diabetic mouse respectively. *p< 0.05 compared to normal.

Article Snippet: Sonicated DNA was immunoprecipitated for 5mC/5hmC using methylated/ hydroxymethylated DNA Immunoprecipitation (MeDIP/hMeDIP) kits (EPIGENTEK, Farmingdale, NY).

Techniques: Binding Assay, Methylation, Immunoprecipitation, Control, SYBR Green Assay, Amplification

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Dynamic DNA methylation of matrix metalloproteinase-9 in the development of diabetic retinopathy

doi: 10.1038/labinvest.2016.78

Figure Lengend Snippet: The levels of 5hmC are increased at the retinal MMP-9 promoter in diabetes, and hydroxymethylation enzyme machinery is activated. (a) 5hmC levels were quantified at the promoter region in sonicated DNA using hMeDIP immunoprecipitation kit. The total genomic DNA obtained from normal mouse retina was used as an input DNA control. (b) The enzyme activity of Tet was measured in 10–20μg retina using TET Activity/Inhibition Assay Kit and the values obtained from the normal mouse retina are considered as 100% (c) The gene transcripts of Tet1 , Tet2 and Tet3 were quantified SYBR green-based q-PCR, and the protein expression of Tet2 was determined by western blot technique. β-actin was used as a house keeping gene (q-PCR)/loading control (western blot). (d) Binding of Tet2 at the MMP-9 promoter was determined in the Tet2 immunoprecipitated crosslinked retina using IgG as a negative antibody control (indicated as ^). Data are represented as mean ± SD from 6–7 mice in each group, with each measurement made in duplicate. *p< 0.05 compared to normal.

Article Snippet: Sonicated DNA was immunoprecipitated for 5mC/5hmC using methylated/ hydroxymethylated DNA Immunoprecipitation (MeDIP/hMeDIP) kits (EPIGENTEK, Farmingdale, NY).

Techniques: Sonication, Immunoprecipitation, Control, Activity Assay, Inhibition, SYBR Green Assay, Expressing, Western Blot, Binding Assay