Journal: Frontiers in Cell and Developmental Biology

Article Title: Single-Cell RNA Sequencing Reveals the Role of Phosphorylation-Related Genes in Hepatocellular Carcinoma Stem Cells

doi: 10.3389/fcell.2021.734287

Figure Lengend Snippet: Immunohistochemistry (IHC) data of AURKA and EZH2 from the Human Protein Atlas. Higher expression of AURKA and EZH2 by immunohistochemistry in HCC compared with normal tissue.

Article Snippet: The EZH2 inhibitor gambogenic acid was purchased from MCE (Shanghai, China).

Techniques: Immunohistochemistry, Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Single-Cell RNA Sequencing Reveals the Role of Phosphorylation-Related Genes in Hepatocellular Carcinoma Stem Cells

doi: 10.3389/fcell.2021.734287

Figure Lengend Snippet: Gambogenic acid (EZH2 inhibitor) and alisertib (AURKA inhibitor) inhibit HCC cell proliferation, migration, and invasion. (A) Hep3B and Huh7 cells were treated with gambogenic acid (EZH2 inhibitor) or alisertib (AURKA inhibitor) at different concentrations (0–100 μM) for 48 h, and cell viability was determined by Calcein-AM/PI staining assays. (B) Hep3B and Huh7 cells were treated with DMSO (solvents of gambogenic acid and alisertib) at different concentrations (0–100 μM) for 48 h, and cell viability was determined by Calcein-AM/PI staining assays. (C) Hep3B and Huh7 cells were treated with gambogenic acid (EZH2 inhibitor, 2 μM) or alisertib (AURKA inhibitor, 10 μM) for 0, 12, 24, 32, 48, and 60 h, and cell viability was determined by Calcein-AM/PI staining assays. (D) Colony formation assays were conducted to analyze Hep3B and Huh7 cell proliferation with gambogenic acid (2 μM) or alisertib (10 μM) treatment. ( E-F) Wound healing assays (E) and Transwell assays (F) were performed to detect the cell migratory abilities of Hep3B and Huh7 cells treated with gambogenic acid (2 μM) or alisertib (10 μM). (G) Transwell assays were performed to detect the cell invasion abilities of Hep3B and Huh7 cells treated with gambogenic acid (2 μM) or alisertib (10 μM). Data are expressed as the means ± s.d. Differences were considered statistically significant if p < 0.05. ns, no significance, * p < 0.05, ** p < 0.01, *** p < 0.001. (H, I) Expression of AURKA (H) and EZH2 (I) in TCGA-LIHC based on TP3 mutation status.

Article Snippet: The EZH2 inhibitor gambogenic acid was purchased from MCE (Shanghai, China).

Techniques: Migration, Staining, IF-P, Expressing, Mutagenesis

Journal: Developmental cell

Article Title: MicroRNAs Overcome Cell Fate Barrier by Reducing EZH2-Controlled REST Stability during Neuronal Conversion of Human Adult Fibroblasts

doi: 10.1016/j.devcel.2018.06.007

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit monoclonal anti-EZH2 , Cell Signaling Technology , Cat# 5246S; RRID:AB_10694683.

Techniques: shRNA, Control, ChIP-qPCR, Recombinant, Chromatin Immunoprecipitation, Mutagenesis, Bicinchoninic Acid Protein Assay, Luciferase, Protease Inhibitor, Membrane, Software

Journal: Open Biology

Article Title: HOXD-AS1 confers cisplatin resistance in gastric cancer through epigenetically silencing PDCD4 via recruiting EZH2

doi: 10.1098/rsob.190068

Figure Lengend Snippet: HOXD-AS1 epigenetically inhibited PDCD4 through binding with EZH2 in gastric cancer cells. ( a ) The HOXD-AS1 subcellular location was determined in BGC823/DDP cells. U6 was used as a nucleus control and GAPDH acted as a cytoplasm control. ( b ) PDCD4 protein levels were detected in si-con or si-HOXD-AS1-transfected BGC823/DDP cells. ( c ) EZH2 and PDCD4 protein levels were measured in si-con or si-EZH2-transfected BGC823/DDP cells. The RNA pull-down assay ( d ) and RIP assay ( e ) were performed to identify direct binding between HOXD-AS1 and EZH2. ( f ) ChIP of EZH2 occupancy and H3K27me3 binding in the PDCD4 promoter in BGC823/DDP cells. ( g ) Luciferase reporter assay evaluated the PDCD4 promoter activity in BGC823/DDP cells transfected with (si-HOXD-AS1 or si-con) or (HOXD-AS1 or Vector). * p < 0.05.

Article Snippet: The empty vector pcDNA3.1 (vector) or HOXD-AS1 overexpressing vector pcDNA3.1-HOXD-AS1 (HOXD-AS1) and small interfering RNAs against HOXD-AS1 (si-HOXD-AS1), EZH2 (si-EZH2) or PDCD4 (si-PDCD4) or their scramble negative siRNA (si-con) were obtained from Genepharma (Shanghai, China).

Techniques: Binding Assay, Transfection, Pull Down Assay, Luciferase, Reporter Assay, Activity Assay, Plasmid Preparation

Journal: Cancer discovery

Article Title: Epigenetic reprogramming sensitizes CML stem cells to combined EZH2 and tyrosine kinase inhibition

doi: 10.1158/2159-8290.CD-16-0263

Figure Lengend Snippet: a. Top; the mean fold changes in mRNA expression for 6 PRC2 components and BCR-ABL1 in normal (HSCs and HPCs) and in CML (LSCs and LPCs) cells as determined by Fluidigm analysis performed in triplicate. Fold changes (log2) are expressed relative to levels found in HSCs. Error bars are standard error measurements (SEMs). Bottom; the mean levels of EZH2 mRNA relative to EZH1 mRNA in each of the bioreplicate samples as above. Significant p values (*; Student’s t-test) between equivalent cell type comparisons (HSC vs LSC, HPC vs LPC) are shown. Bioreplicates are n = 4 (HPC), n = 3 (HSC), n = 10 (LSC), n = 10 (LPC). b. Schematic diagram depicts how changes in H3K27me3 levels between normal and CML cells were determined with respect to three pairwise cell type comparisons: HSCs vs HPCs (“norm”), HSCs vs LSCs (“early”), LSCs vs LPCs (“late”). c. Venn diagram depicts the relationship between the numbers of promoters which show altered levels of H3K27me3 in three pairwise comparisons (HSC→HPC; HSC→LSC; LSC→LPC). d. Examples of H3K27me3 read densities across genes that are up- (red arrow) or down-(green arrow) regulated (mRNA) in CML cells compared to HSCs. Location of transcription start site and direction of transcription are shown by →. Profiles were visualised in the UCSC genome browser (hg18; NCBI build 36.1). e. Bar diagrams show the percentages (%) of all reprogrammed promoters having significant gains (red) or losses (green) of H3K27me3 (p < 0.05; one sample t-tests) when their cognate mRNA levels are down- (↓) or up- (↑) regulated (Affymetrix analysis) in each of three pairwise comparisons for n = 3 bioreplicate CML(1,2,3) and normal samples. f. Bar diagrams show the percentages (%) of all promoters reprogrammed in all three CML samples (common) having significant gains (red) or losses (green) of H3K27me3 (p < 0.05; one sample t-tests) when their cognate mRNA levels are down- (↓) or up- (↑) regulated (Affymetrix analysis) as above. In e and f, expected percentages of H3K27me3 gains associated with global reprogramming of H3K27me3 levels, irrespective of mRNA expression changes, are shown (white bars). Significant p values (*) are shown based on observed vs expected levels of H3K27me3 gains or losses at promoters (Yates Chi-squared or Fischer Exact tests). g. Schematic representation of H3K27me3 reprogramming in CML leading to increased mechanistic coupling of H3K27me3 levels and mRNA expression - consistent with an altered dependency for EZH2 in CML (upward green arrow). HSCs require EZH1(14) but not EZH2(15) (downward green arrow).

Article Snippet: EZH2 knockdown EZH2 shRNAs were subcloned from the pLKO.1 puro vector (Sigma Mission shRNA collection) into the Nde I and Spe I sites of the pLKO.1 GFP vector.

Techniques: Expressing

Journal: Cancer discovery

Article Title: Epigenetic reprogramming sensitizes CML stem cells to combined EZH2 and tyrosine kinase inhibition

doi: 10.1158/2159-8290.CD-16-0263

Figure Lengend Snippet: a. Schematic for EZH2i treatment in primary CML and normal CD34+ cells. n = 3 bioreplicates in all instances described below. b. Representative western analysis of H3K27me3 and H3K27me1 levels in CML and normal CD34+ cells treated for 6 days with increasing concentrations of EZH2i as indicated. L = laemmli buffer; total cellular protein. A = acid extraction; enriched for histones. Levels of bulk histone H3 were used as loading controls. c. Viability of CML and normal CD34+ cells treated with EZH2i for 6 days. Mean total viable cell counts (top); mean viable undivided (CTVmax) CD34+ cells (bottom). d. Levels of apoptosis (Annexin V-positive cells) in CML and normal CD34+ cells treated with EZH2i. Mean total apoptosis (top) and mean apoptosis in undivided (CTVmax) CD34+ cells (bottom). e. Colonogenic (CFC) potential of CML and normal CD34+ cells treated with EZH2i. Mean total CFC outputs (top) and GEMM outputs (bottom) from CD34+ cells; total CFC outputs from LSCs (CD34+CD38-) (bottom). f. Colonogenic potential of CML and normal CD34+ cells from LTC-IC after 3 days or 6 days treatment with EZH2i. % of cells Ph+ (i.e., BCR-ABL+) in NDC (day 6) is shown by the arrowhead (red). g. Effect of EZH2 shRNA knockdown in CML34+ cells. Cell counts (left), CFC output (middle) and apoptosis (Annexin V-positive cells) (right) for CML CD34+ cells transduced with EZH2 or control shRNA for 3 days. Two different EZH2 shRNAs were used to derive data. h. Scatterplot depicting global mRNA expression changes (Z-scores) for 975 and 1473 genes in CML and normal CD34+ cells respectively as a consequence of EZH2i treatment (and where both mRNA and H3K27me3 status is known) (E-MTAB-2893; E-MTAB-3552). H3K27me3 targets (> +/- 1 SD from the mean) are highlighted (red). Significant p values (*; one sample t-tests in c → g) are shown in the key below panel g. EZH2i is GSK343 for data shown in b → f and in h. i. Venn diagrams depict the degree of overlap between genes showing mRNA changes due to EZH2i treatment in CML and normal CD34+ cells; all genes (top), H3K27me3 target genes (bottom). % of overlap with respect to totals for CML CD34+ cells (left).

Article Snippet: EZH2 knockdown EZH2 shRNAs were subcloned from the pLKO.1 puro vector (Sigma Mission shRNA collection) into the Nde I and Spe I sites of the pLKO.1 GFP vector.

Techniques: Western Blot, shRNA, Transduction, Expressing

Journal: Cancer discovery

Article Title: Epigenetic reprogramming sensitizes CML stem cells to combined EZH2 and tyrosine kinase inhibition

doi: 10.1158/2159-8290.CD-16-0263

Figure Lengend Snippet: a. Summary of global mean mRNA expression changes found in LSCs after treatment with TKI for 7 days in vitro based on Affymetrix analysis (E-MTAB-2594). Percentages of genes up- or down-regulated are shown for H3K27me3 targets and other genes for each TKI treatment (n = 6 bioreplicates in all cases). Total number of mRNAs affected shown along the bottom. b. Representative western analysis (L = laemmli buffer) of H3K27me3 and H3K27me1 levels in CML CD34+ treated with DAS with and without increasing concentrations of EZH2i (GSK343). c. Viability of CML CD34+ cells treated with EZH2i and DAS for 6 days. Mean total viable cell counts (left); mean viable undivided (CTVmax) CD34+ cells (right). d. Levels of apoptosis (Annexin V-positive cells) in CML CD34+ cells treated with EZH2i and DAS for 6 days. Mean total apoptosis (left) and mean apoptosis in undivided (CTVmax) CD34+ cells (right). e. Colonogenic (CFC) potential of CML cells treated with EZH2i and DAS for 6 days. Mean total CFC outputs (left) and GEMM outputs (middle) from CML CD34+ cells and total CFC outputs from LSCs (CD34+CD38-) (right). f. Colonogenic potential of CML CD34+ cells from LTC-IC following 3 or 6 day treatment with EZH2i and DAS (same samples as shown in Fig. 3e). Expected levels (green dotted line) are based on efficacy of EZH2i alone on CD34+ cells (from Fig. 3f). g. Cell counts (left) and CFC output (right) for CML CD34+ cells transduced with EZH2 or control shRNA for 4 days in the presence of DAS. Two different EZH2 shRNAs were used to derive data. Results in c → g are shown as a percentage change from treatment with DAS (150 nM) only, unless indicated. n = 3 bioreplicates in c,d,e,f; n = 4 bioreplicates in g. h. Schematic showing the experimental design for EZH2i and TKI treatment of NSG mice engrafted with CML CD34+ samples. i. Representative FACs scatter plots showing the proportions (%) of human CML CD45+ cells staining positive for CD34+ (hCD34+) in bone marrow from NSG xenografts following in vivo treatment with EZH2i (EPZ-6438) or TKI (nilotinib; NIL) alone and in combination for 25 days. SSC = side scatter. j. Left side, mean absolute number (top) and mean % (bottom) of Ph+ hCD45+ cells from bone marrow of NSG xenografts treated with vehicle (NDC), EZH2i, NIL (nilotinib) and EZH2i + NIL (25 day treatment; n = 2 CML CD34+ samples; n = 8 mice per arm). Right side, mean absolute number (top) and mean % (bottom) Ph+ hCD45+CD34+ cells from the same experiments. k. Mean absolute number (top) and mean % (bottom) of Ph+ hCD45+CD34+CD38- cells from bone marrow of NSG xenografts treated as above (n = 1 CML CD34+ sample; n = 3-4 mice per arm). In j, k, % Ph+ cells are expressed as a % of hCD45+ in NDC. Significant p values (*; one sample t-tests in c → g; Student t-tests in bar diagrams of j, k) are as shown in the key next to panel k.

Article Snippet: EZH2 knockdown EZH2 shRNAs were subcloned from the pLKO.1 puro vector (Sigma Mission shRNA collection) into the Nde I and Spe I sites of the pLKO.1 GFP vector.

Techniques: Expressing, In Vitro, Western Blot, Transduction, shRNA, Staining, In Vivo

Journal: Cancer discovery

Article Title: Epigenetic reprogramming sensitizes CML stem cells to combined EZH2 and tyrosine kinase inhibition

doi: 10.1158/2159-8290.CD-16-0263

Figure Lengend Snippet: a. Main panel, heat maps showing mean fold changes in the mRNA expression (Fluidigm) of a number of apoptotic pathway genes following treatment of CML CD34+ samples with EZH2i (1000 nM) or DAS (150 nM) alone or in combination (n =4 bioreplicates assayed in triplicate); mean changes in mRNA expression of specific p53 pathway genes and BCL6 are shown (right). b. Bar diagrams showing comparisons of predicted (from individual treatments using an additive model; grey) and actual changes in mean mRNA expression levels of H3K27me3 (blue; left) and other targets (pink; right) following treatment with both EZH2i and DAS in each bioreplicate CML sample (n = 4) shown in a. Error bars are SEMs in all instances. Significant p values (*; one sample t-tests in a, Student t-tests in bar diagrams of b) are as shown in the key next to panel b. c. Representative western analysis (laemmli buffer) of p53 levels in two CML CD34+ samples before and after EZH2i +/- DAS treatment. d. Schematic of a putative interactome map between EZH2/H3K27me3 (blue), BCL6 (red) and p53 (purple) targets which mediate cell cycle arrest and apoptosis in CML CD34+ cells. BCL6 targets were obtained from previously published data(22). BCL6 is self-regulatory as shown. Cell cycle inhibitors and activators are shown in orange and green respectively.

Article Snippet: EZH2 knockdown EZH2 shRNAs were subcloned from the pLKO.1 puro vector (Sigma Mission shRNA collection) into the Nde I and Spe I sites of the pLKO.1 GFP vector.

Techniques: Expressing, Western Blot

Journal: Investigative Ophthalmology & Visual Science

Article Title: Crosstalk Between Histone and DNA Methylation in Regulation of Retinal Matrix Metalloproteinase-9 in Diabetes

doi: 10.1167/iovs.17-22706

Figure Lengend Snippet: Effect of high glucose on trimethylation of H3K27 and its regulation by Ezh2. HRECs incubated in high glucose for 4 days in the presence or absence of DZNep (5 μM), or transfected with Ezh2-siRNA, were analyzed for (a) H3K27me3 levels at the MMP-9 promoter by immunoprecipitating H3K27me3 in the cross-linked samples, followed by PCR using the primers for the AP-1 binding region of the MMP-9 promoter. IgG (^) was used as an antibody control. Ct values were normalized with the values from input by delta delta Ct method. (b) Agarose gel picture showing the band intensity. Values are represented as mean ± SD from three different cell preparations; 5 and 20 mM = 5- and 20-mM glucose, respectively; 5 + D and 20 + D = cells in 5- and 20-mM glucose, respectively, with DZNep; 20+E-si and 20+SC = Ezh2-siRNA or scrambled RNA cells in 20-mM glucose; 5 + E-si = Ezh2-siRNA transfected cells in 5-mM glucose; Mann = 20-mM mannitol. *P < 0.05 compared with 5-mM glucose, #P < 0.05 compared with 20-mM glucose.

Article Snippet: The specificity of the assay was validated by resolving the PCR products in 2% agarose gel., Histone methyltransferase, Ezh2, activity was measured in nuclear fraction prepared following the manufacturer's instruction (EPIGENTEK, Farmingdale, NY, USA).

Techniques: Incubation, Transfection, Binding Assay, Control, Agarose Gel Electrophoresis

Journal: Investigative Ophthalmology & Visual Science

Article Title: Crosstalk Between Histone and DNA Methylation in Regulation of Retinal Matrix Metalloproteinase-9 in Diabetes

doi: 10.1167/iovs.17-22706

Figure Lengend Snippet: Effect of high glucose on Ezh2: (a) Ezh2 mRNA levels were quantified in the cDNA by real time PCR using β-actin as the housekeeping gene. (b) The enzyme activity of Ezh2 was determined using Ezh2 activity/inhibition assay kit, and the values obtained from cells in 5-mM glucose are considered as 100%. Values are mean ± SD of three to four experiments, each done in duplicate. *P < 0.05 compared with 5-mM glucose, #P < 0.05 compared with 20-mM glucose.

Article Snippet: The specificity of the assay was validated by resolving the PCR products in 2% agarose gel., Histone methyltransferase, Ezh2, activity was measured in nuclear fraction prepared following the manufacturer's instruction (EPIGENTEK, Farmingdale, NY, USA).

Techniques: Real-time Polymerase Chain Reaction, Activity Assay, Inhibition

Journal: Investigative Ophthalmology & Visual Science

Article Title: Crosstalk Between Histone and DNA Methylation in Regulation of Retinal Matrix Metalloproteinase-9 in Diabetes

doi: 10.1167/iovs.17-22706

Figure Lengend Snippet: Effect of high glucose on Ezh2 recruitment at the MMP-9 promoter. The binding of Ezh2 at the MMP-9 promoter was quantified using Ezh2 monoclonal antibody, (a) followed by amplification of the promoter region by real time qPCR. (b) Product sizes were confirmed on 2% agarose gel. *P < 0.05 compared with 5-mM glucose; #P < 0.05 compared with 20-mM glucose.

Article Snippet: The specificity of the assay was validated by resolving the PCR products in 2% agarose gel., Histone methyltransferase, Ezh2, activity was measured in nuclear fraction prepared following the manufacturer's instruction (EPIGENTEK, Farmingdale, NY, USA).

Techniques: Binding Assay, Amplification, Agarose Gel Electrophoresis

Journal: Investigative Ophthalmology & Visual Science

Article Title: Crosstalk Between Histone and DNA Methylation in Regulation of Retinal Matrix Metalloproteinase-9 in Diabetes

doi: 10.1167/iovs.17-22706

Figure Lengend Snippet: Effect of regulation of Ezh2 on MMP-9: (a) mRNA levels of MMP-9 were measured using real time qPCR, and each measurement was made in duplicate in three to five samples in each group. (b) Enzyme activity of MMP-9 was quantified in by an ELISA method using a fluorescence kit. (c) Mitochondrial localization of MMP-9 was determined by immunofluorescence using DyLight 488-conjugated secondary antibody for MMP-9 and Texas red-conjugated for CoxIV. *P < 0.05 compared with 5-mM glucose; #P < 0.05 compared with 20-mM glucose.

Article Snippet: The specificity of the assay was validated by resolving the PCR products in 2% agarose gel., Histone methyltransferase, Ezh2, activity was measured in nuclear fraction prepared following the manufacturer's instruction (EPIGENTEK, Farmingdale, NY, USA).

Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay, Fluorescence, Immunofluorescence

Journal: Investigative Ophthalmology & Visual Science

Article Title: Crosstalk Between Histone and DNA Methylation in Regulation of Retinal Matrix Metalloproteinase-9 in Diabetes

doi: 10.1167/iovs.17-22706

Figure Lengend Snippet: Ezh2 regulates DNA methylation of MMP-9 promoter. Recruitment of (a) Dnmt1 and (b) Tet2 at the MMP-9 promoter was measured in the cells incubated in high glucose ± DZNep, or transfected with Ezh2-siRNA or scrambled RNA (SC) by ChIP technique. (c) A representative agarose gel showing the accompanying band intensities. (d) The levels of 5hmC were quantified using hMeDIP immunoprecipitation kit. *P < 0.05 compared with 5-mM glucose; #P < 0.05 compared with 20-mM glucose.

Article Snippet: The specificity of the assay was validated by resolving the PCR products in 2% agarose gel., Histone methyltransferase, Ezh2, activity was measured in nuclear fraction prepared following the manufacturer's instruction (EPIGENTEK, Farmingdale, NY, USA).

Techniques: DNA Methylation Assay, Incubation, Transfection, Agarose Gel Electrophoresis, Immunoprecipitation

Journal: Investigative Ophthalmology & Visual Science

Article Title: Crosstalk Between Histone and DNA Methylation in Regulation of Retinal Matrix Metalloproteinase-9 in Diabetes

doi: 10.1167/iovs.17-22706

Figure Lengend Snippet: Regulation of Ezh2 activation prevents glucose-induced decrease in mtDNA transcription and cell apoptosis. (a) mRNA levels of mtDNA-encoded Cytb were quantified by real time qPCR using β-actin as the housekeeping gene. (b) Cell apoptosis was determined using an ELISA kit for histone-associated-DNA-fragments. The values are represented as mean ± SD from three to four samples/group, each measurement made in duplicate. *P < 0.05 compared with 5-mM glucose; #P < 0.05 compared with 20-mM glucose.

Article Snippet: The specificity of the assay was validated by resolving the PCR products in 2% agarose gel., Histone methyltransferase, Ezh2, activity was measured in nuclear fraction prepared following the manufacturer's instruction (EPIGENTEK, Farmingdale, NY, USA).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay

Journal: Investigative Ophthalmology & Visual Science

Article Title: Crosstalk Between Histone and DNA Methylation in Regulation of Retinal Matrix Metalloproteinase-9 in Diabetes

doi: 10.1167/iovs.17-22706

Figure Lengend Snippet: Diabetes increases H3K27me3 and Ezh2 recruitment at the MMP-9 promoter. Rat retinal microvessels were analyzed for (a) H3K27me3 and Ezh2 binding at the MMP-9 promoter by ChIP technique using IgG (^) as an antibody control. (b) Representative picture of an agarose gel showing the band intensities (c) Ezh2 mRNA levels were quantified in the cDNA by real time qPCR, and β-actin was used as a housekeeping gene. Values are represented as mean ± SD obtained from five to seven rats in each group. Norm and Diab = normal and diabetic rats, respectively. *P < 0.05 compared with normal.

Article Snippet: The specificity of the assay was validated by resolving the PCR products in 2% agarose gel., Histone methyltransferase, Ezh2, activity was measured in nuclear fraction prepared following the manufacturer's instruction (EPIGENTEK, Farmingdale, NY, USA).

Techniques: Binding Assay, Control, Agarose Gel Electrophoresis

Journal: Investigative Ophthalmology & Visual Science

Article Title: Crosstalk Between Histone and DNA Methylation in Regulation of Retinal Matrix Metalloproteinase-9 in Diabetes

doi: 10.1167/iovs.17-22706

Figure Lengend Snippet: Retinal microvessels from human donors with diabetic retinopathy have increased H3K27me3 and Ezh2 recruitment at their MMP-9 promoter. (a) H3K27me3 and recruitment of (b) Ezh2 or (c) Dnmt1/Tet2 was quantified by ChIP technique using specific antibodies, and 5hmC by hMeDIP immunoprecipitation kit. (d) MMP-9 mRNA was quantified by real-time qPCR using gene-specific primers, in the retinal microvessels from human donors with 10 to 43 years of diabetes and documented retinopathy (diabetes), and their age-matched nondiabetic donors (normal). Amplification of the promoter region of MMP-9 was performed by qPCR, and the product sizes were confirmed on 2% agarose gel. Values are represented as mean ± SD; *P < 0.05 versus nondiabetic.

Article Snippet: The specificity of the assay was validated by resolving the PCR products in 2% agarose gel., Histone methyltransferase, Ezh2, activity was measured in nuclear fraction prepared following the manufacturer's instruction (EPIGENTEK, Farmingdale, NY, USA).

Techniques: Immunoprecipitation, Amplification, Agarose Gel Electrophoresis