Journal: iScience

Article Title: Neonatal intake of Omega-3 fatty acids enhances lipid oxidation in adipocyte precursors

doi: 10.1016/j.isci.2022.105750

Figure Lengend Snippet: Bulk RNA-seq of flow-sorted APC CD24 + progenitors and CD24 − preadipocytes reveals transcriptomic signatures for adipogenic, mitochondrial, and energetics pathways by low n6/n3 FA ratios (A) Differential gene expression by n6/n3 exposure group within either progenitor or preadipocyte APC subtypes was analyzed using comparative analysis in Ingenuity Pathway Analysis. Significantly enriched canonical pathways in common between CD24 + progenitor and CD24 − preadipocyte APCs were ordered by FDR p value (≤0.01) and colored by the predicted activation Z score. Coloring indicates common pathways predicted to be “on” (yellow), “off” (navy), or “no prediction” (white) due to low n6/n3 exposure. Pathway activation Z score values are presented for either APC subtype, and values ≥2.0 or ≤ −2.0 are considered significant. (B) Significantly different genes in either CD24 + progenitor or CD24 − preadipocyte relative to the high n6/n3 FA ratio group colored by Log2 fold change and grouped into transcription factor genes (Regulatory) and signal transduction pathway genes (Ligands/Kinases). (C) Significantly different lipid metabolism, mitochondrial, and glycolytic genes indicating that the low n6/n3 FA ratio changes oxidative capacity potentially through aldehyde dehydrogenases (Aldh1a1 and Aldh1a3), uncoupling protein 2 (Ucp2), citrate synthase (CS), isocitrate dehydrogenase (Idh1 and 2), malic enzyme (Me1), malate dehydrogenase (Mdh1), phosphofructokinases (Pfkm, Pfkp, and Pfkfb1), and transketolase (Tk) (See also Figure S2 ). (D) Canonical Pathway enrichment within either CD24 + progenitor or CD24 − preadipocyte APCs plotted by the percentage DE-Gs within a pathway (%) by the pathway enrichment significance (-log(p value)) and colored by the predicted pathway activation ( Z score, >2.0 and < −2.0 was considered significant) (See also for all pathways). (E) Immunoblots for NR2F2, UCP2, C/EBPα, and DNMT1 from all lineage negative, CD29+/CD34+, and Sca1+ APCs isolated by flow cytometry from pooled litters (n = 3 litter dyads per n6/n3 FA ratio group; litters standardized to 6-8 pups/litter). (F) EVC005 murine APC cell line was treated with and without NR2F2 ligand 1-DSO for 48 h, which induced gene expression of Prdm16, P2rx5, Runx1, Ucp2, Dnmt1, Pparγ2, and C/ebpα. (G) TMRE live cell mitochondrial potential dye following 48 h of 1-DSO treatment in EVC005 cells, indicating an increased mitochondrial potential. Panels F and G are n = 3 wells per treatment group. Scale bar represents 50 μm. Data are represented as mean ± SEM.

Article Snippet: Dnmt1 mouse TaqMan Primer , ThermoFisher Scientific , Cat# Mm01151063_m1.

Techniques: RNA Sequencing, Gene Expression, Activation Assay, Transduction, Western Blot, Isolation, Flow Cytometry

Journal: iScience

Article Title: Neonatal intake of Omega-3 fatty acids enhances lipid oxidation in adipocyte precursors

doi: 10.1016/j.isci.2022.105750

Figure Lengend Snippet:

Article Snippet: Dnmt1 mouse TaqMan Primer , ThermoFisher Scientific , Cat# Mm01151063_m1.

Techniques: Recombinant, High Molecular Weight, Enzyme-linked Immunosorbent Assay, Software, Modification

Journal: Investigative Ophthalmology & Visual Science

Article Title: Epigenetic Modifications Compromise Mitochondrial DNA Quality Control in the Development of Diabetic Retinopathy

doi: 10.1167/iovs.19-27602

Figure Lengend Snippet: Effect of high glucose on DNA methylation of Mlh1 promoter and its transcriptional regulation. HRECs, incubated in high glucose, were analyzed for (a) 5mC levels at Mlh1 promoter by methylated DNA immunoprecipitation method, (b, c) Dnmt1 and Sp1 binding by ChIP technique, and (d) Mlh1 gene transcripts by qPCR. Each measurement was made in duplicate in three to four samples per group. The values obtained from cells in NG are considered as 1. NG and HG = 5 mM and 20 mM glucose, respectively; D-si and SC = cells transfected with Dnmt1-siRNA and control negative RNA, respectively, and incubated in HG; Aza = 1 μM Aza-2′-deoxyxytidine. *P < 0.05 versus NG and #P < 0.05 versus HG.

Article Snippet: L-glucose (20 mM), instead of 20 mM D-glucose, was used as the osmotic/metabolic control., , A group of cells from the fifth to sixth passage were transfected with Dnmt1- siRNA (cat. no. SC-35204, Santa Cruz Biotechnology, Santa Cruz, CA, USA) using transfection reagent (cat. no. SC-29528, Santa Cruz Biotechnology).

Techniques: DNA Methylation Assay, Incubation, Methylation, Immunoprecipitation, Binding Assay, Transfection, Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: Epigenetic Modifications Compromise Mitochondrial DNA Quality Control in the Development of Diabetic Retinopathy

doi: 10.1167/iovs.19-27602

Figure Lengend Snippet: Regulation of DNA methylation and base mismatches. DNA from HRECs was amplified using semi-qPCR for the D-loop and digested with mismatch-specific surveyor endonuclease. The samples were analyzed on a 2% agarose gel. The parent amplicon band intensity was quantified by densitometry, and the values obtained from cells in NG was considered as 100%. Data are represented as mean ± SD, and each measurement was made in duplicate in three to five samples in each group. Mismatches and the parent amplicon band intensity from (a) Dnmt1-siRNA transfected HRECs and (b) Aza-treated HRECs. NG and HG = HRECs in 5 mM and 20 mM glucose, respectively; D-si and SC = cells transfected with Dnmt1-siRNA and negative control RNA, respectively; Aza = 1 μM 5-Aza-2′-deoxyxytidine. *P < 0.05 compared with NG; #P < 0.05 compared with HG.

Article Snippet: L-glucose (20 mM), instead of 20 mM D-glucose, was used as the osmotic/metabolic control., , A group of cells from the fifth to sixth passage were transfected with Dnmt1- siRNA (cat. no. SC-35204, Santa Cruz Biotechnology, Santa Cruz, CA, USA) using transfection reagent (cat. no. SC-29528, Santa Cruz Biotechnology).

Techniques: DNA Methylation Assay, Amplification, Agarose Gel Electrophoresis, Transfection, Negative Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: Epigenetic Modifications Compromise Mitochondrial DNA Quality Control in the Development of Diabetic Retinopathy

doi: 10.1167/iovs.19-27602

Figure Lengend Snippet: Differential DNA methylation of the Mlh1 promoter in HRECs incubated in high glucose. Four CpG-rich regions of the proximal promoter of Mlh1 (−659 to 72; R1-R4) were analyzed for (a) 5mC levels by methylated DNA immunoprecipitation method and (b) Dnmt1 binding by ChIP using IgG (represented as ^) as an antibody control. The values are represented as fold change, and the values obtained from the cells in NG are considered as one. NG and HG = 5 mM and 20 mM glucose, respectively; Aza = 1μM Aza-2′-deoxycytidine. *P < 0.05 compared to NG.

Article Snippet: L-glucose (20 mM), instead of 20 mM D-glucose, was used as the osmotic/metabolic control., , A group of cells from the fifth to sixth passage were transfected with Dnmt1- siRNA (cat. no. SC-35204, Santa Cruz Biotechnology, Santa Cruz, CA, USA) using transfection reagent (cat. no. SC-29528, Santa Cruz Biotechnology).

Techniques: DNA Methylation Assay, Incubation, Methylation, Immunoprecipitation, Binding Assay, Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: Epigenetic Modifications Compromise Mitochondrial DNA Quality Control in the Development of Diabetic Retinopathy

doi: 10.1167/iovs.19-27602

Figure Lengend Snippet: DNA methylation of the Mlh1 promoter in diabetes and its regulation by Dnmt inhibitors. Retinal microvessels from diabetic mice receiving intravitreal administration of Dnmt1-siRNA (D-si) or intraperitoneal injection of Aza-2′-deoxyxytidine (Aza) were analyzed for (a) Mlh1 mRNA levels by qPCR and (b) 5mC levels at the promoter region of Mlh1 by a methylated DNA immunoprecipitation kit. Values are calculated as fold change as compared to normal and are represented as mean ± SD. Nor and Diab = nondiabetic control and streptozotocin-induced diabetic mice, respectively. *P < 0.05 compared to normal and #P < 0.05 compared to diabetes.

Article Snippet: L-glucose (20 mM), instead of 20 mM D-glucose, was used as the osmotic/metabolic control., , A group of cells from the fifth to sixth passage were transfected with Dnmt1- siRNA (cat. no. SC-35204, Santa Cruz Biotechnology, Santa Cruz, CA, USA) using transfection reagent (cat. no. SC-29528, Santa Cruz Biotechnology).

Techniques: DNA Methylation Assay, Injection, Methylation, Immunoprecipitation, Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: Epigenetic Modifications Compromise Mitochondrial DNA Quality Control in the Development of Diabetic Retinopathy

doi: 10.1167/iovs.19-27602

Figure Lengend Snippet: Methylation of MLH1 in human donors with diabetic retinopathy. Retinal microvessels from human donors with documented diabetic retinopathy (DR) and age-matched nondiabetic donors (Norm) were analyzed for MLH1 and DNMT1 gene transcripts by qPCR using β-actin as a housekeeping gene (a). The MLH1 promoter was analyzed for (b) 5mC levels using a methylated DNA immunoprecipitation method and (c) Dnmt1 binding by ChIP technique. Values are mean ± SD of at least six donors in each group, with each measurement made in duplicate, and are presented as fold change compared to nondiabetic donors. ^ IgG antibody control. *P < 0.05 compared to nondiabetic control.

Article Snippet: L-glucose (20 mM), instead of 20 mM D-glucose, was used as the osmotic/metabolic control., , A group of cells from the fifth to sixth passage were transfected with Dnmt1- siRNA (cat. no. SC-35204, Santa Cruz Biotechnology, Santa Cruz, CA, USA) using transfection reagent (cat. no. SC-29528, Santa Cruz Biotechnology).

Techniques: Methylation, Immunoprecipitation, Binding Assay, Control

Journal: The Journal of Clinical Investigation

Article Title: AMPK is necessary for Treg functional adaptation to microenvironmental stress during malignancy and viral pneumonia

doi: 10.1172/JCI179572

Figure Lengend Snippet: ( A – C ) CpG methylation of all gene promoters ( A ), gene promoters of cluster 1 genes identified by k -means clustering of the RNA-seq shown in ( B ), and the Ppargc1a promoter ( C ) in tumor-infiltrating CD4 + Foxp3 YFP+ cells ( n = 4 Prkaa1/2 wt/wt Foxp3 YFP–Cre or control, n = 2 Prkaa1/2 fl/fl Foxp3 YFP–Cre ) and splenic CD4 + Foxp3 YFP+ cells at homeostasis ( n = 3 control, n = 3 Prkaa1/2 fl/fl Foxp3 YFP–Cre ) ( D ) DNMT1 protein expression of splenic CD4 + Foxp3 YFP+ (Treg) cells at homeostasis ( n = 7 control, n = 7 Prkaa1/2 fl/fl Foxp3 YFP–Cre ). 3 independent experiments are shown. DNMT1 peak intensity area was normalized to the corresponding sample’s β-actin peak intensity area. ( E ) Dnmt1 gene expression of splenic CD4 + Foxp3 YFP+ cells at homeostasis ( n = 4 control, n = 4 Prkaa1/2 fl/fl Foxp3 YFP–Cre ) as measured by RNA-seq shown in Figure 1. ( F ) Anti-AMPKα1 and isotype control immunoprecipitates from ex vivo induced (i)Treg cell lysates blotted for DNMT1 protein. Independent biological replicates are shown. ( G ) Representative microscopy images of AMPKα-sufficient iTreg cells showing AMPKα1 and DNMT1 subcellular localization. Scale bars: 5 μm. ( H ) MitoTracker Deep Red (MitoTracker DR) mean fluorescence intensity (MFI) of AMPKα-sufficient (control) and -deficient splenic CD4 + Foxp3 YFP+ cells treated with either vehicle ( n = 8 control, n = 10 Prkaa1/2 fl/fl Foxp3 YFP–Cre ), 50 nM decitabine (DAC, n = 7 control, n = 7 Prkaa1/2 fl/fl Foxp3 YFP–Cre ), or 100 nM DAC ( n = 7 control, n = 7 Prkaa1/2 fl/fl Foxp3 YFP–Cre ). * P or q < 0.05, NS, not significant according to Mann-Whitney U test ( D and E ) with 2-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli with Q = 5% ( H ).

Article Snippet: Cell lysates were subjected to gel electrophoresis and transferred to membranes that were incubated with an antibody against AMPKα1 (Abcam cat. no. ab32047), DNMT1 (Cell Signaling cat. no. 5032), and β-actin (Abcam cat. no. ab8227) overnight at 4°C with constant agitation.

Techniques: CpG Methylation Assay, RNA Sequencing, Control, Expressing, Gene Expression, Ex Vivo, Microscopy, Fluorescence, MANN-WHITNEY

Journal: PloS one

Article Title: Cell cycle-dependent turnover of 5-hydroxymethyl cytosine in mouse embryonic stem cells.

doi: 10.1371/journal.pone.0082961

Figure Lengend Snippet: Figure 1. 5hmC content is diluted during replication. A. Hemi-hydroxymethylated DNA (CG/5hmCG) is not a good substrate for Dnmt1. The DNA methylation activity of mouse Dnmt1, Dnmt3a, and Dnmt3b towards 35-bp unmethylated (CG/CG), hemi- methylated (CG/5mCG), or hemi-hydroxymethylated (CG/5hmCG) DNA was determined. B. Gel mobility shift assaying of the SRA domain of mouse Uhrf1. The indicated concentrations of SRA were incubated with either 12-bp CG/5mC, CG/5hmCG, or CG/CG, followed by electrophoresis (left panel). The complex of the SRA and 32P-labeled CG/5mCG was competed with the indicated amounts of non-labeled CG/5mCG, CG/5hmCG, or CG/CG DNA (right panel). DNA bound to SRA (B) and free DNA (F) are indicated.

Article Snippet: Solubilized chromatin was incubated with mouse monoclonal IgG (Cat No. 12-371, Millipore), rabbit IgG (Cat No. 12-370, Millipore), anti-Dnmt1 mouse monoclonal Dnmt1 (clone: 60B1220.1, Cat No. IMG-261A, Imgenex), anti-Dnmt3a/3a2 [5], anti-Dnmt3b [2], anti-Tet1 (Cat. No. 09-872, Millipore), antiTet2 (Cat No. R1086-6b, Abiocode), or anti-Tet3 (Cat. No. 61395, Activemotif) antibodies at 4°C overnight.

Techniques: DNA Methylation Assay, Activity Assay, Methylation, Mobility Shift, Incubation, Electrophoresis, Labeling

Journal: PloS one

Article Title: Cell cycle-dependent turnover of 5-hydroxymethyl cytosine in mouse embryonic stem cells.

doi: 10.1371/journal.pone.0082961

Figure Lengend Snippet: Figure 3. Dnmt3a and Dnmt3b mainly provide 5mC for the hydroxymethylation in mESCs. The 5mC, 5hmC, and Tet mRNA contents of J1 parent, Dnmt1 (1-KO), Dnmt3a and Dnmt3b (3-DKO), Dnmt3a (3a-KO), Dnmt3b (3b-KO), and Dnmt1, Dnmt3a and Dnmt3b (TKO) knockout mESCs, and ectopically expressed TAP-tagged Dnmt3a (3a-TAP) or Dnmt3a2 (3a2-TAP) in 3-DKO mESCs were determined. A. The 5mC contents (%) were determined as M.SssI methylation ability from a standard curve (Figure S1A). B. The 5hmC contents were determined from the standard curve obtained on β-GT assaying (Figure S1B). The values are the averages ± SD determined for three independent genomic DNA samples. C. Relative mRNA expression of Tet1, Tet2, and Tet3 was evaluated by semi-quantitative RT-PCR. (-) indicates the product of PCR without the template.

Article Snippet: Solubilized chromatin was incubated with mouse monoclonal IgG (Cat No. 12-371, Millipore), rabbit IgG (Cat No. 12-370, Millipore), anti-Dnmt1 mouse monoclonal Dnmt1 (clone: 60B1220.1, Cat No. IMG-261A, Imgenex), anti-Dnmt3a/3a2 [5], anti-Dnmt3b [2], anti-Tet1 (Cat. No. 09-872, Millipore), antiTet2 (Cat No. R1086-6b, Abiocode), or anti-Tet3 (Cat. No. 61395, Activemotif) antibodies at 4°C overnight.

Techniques: Knock-Out, Methylation, Expressing, Quantitative RT-PCR

Journal: PloS one

Article Title: Cell cycle-dependent turnover of 5-hydroxymethyl cytosine in mouse embryonic stem cells.

doi: 10.1371/journal.pone.0082961

Figure Lengend Snippet: Figure 5. Dnmt3a and Dnmt3b-dependent 5mC are responsible for the production of 5hmC. The 5hmC (A) and 5mC (B) contents of J1 (blue bars), Dnmt1 (1-KO, red bars), and Dnmt3a and Dnmt3b (3-DKO, light green bars) knockout mESCs were determined by q-PCR in the promoters of five representative 5hmC-enriched genes. The values are the averages + SD determined for three independent genomic DNA samples.

Article Snippet: Solubilized chromatin was incubated with mouse monoclonal IgG (Cat No. 12-371, Millipore), rabbit IgG (Cat No. 12-370, Millipore), anti-Dnmt1 mouse monoclonal Dnmt1 (clone: 60B1220.1, Cat No. IMG-261A, Imgenex), anti-Dnmt3a/3a2 [5], anti-Dnmt3b [2], anti-Tet1 (Cat. No. 09-872, Millipore), antiTet2 (Cat No. R1086-6b, Abiocode), or anti-Tet3 (Cat. No. 61395, Activemotif) antibodies at 4°C overnight.

Techniques: Knock-Out

Journal: PloS one

Article Title: Cell cycle-dependent turnover of 5-hydroxymethyl cytosine in mouse embryonic stem cells.

doi: 10.1371/journal.pone.0082961

Figure Lengend Snippet: Figure 6. Dnmt1, Dnmt3a, Dnmt3b, and Tet1 are recruited to 5hmC-enriched promoters. The occupancy of Dnmt1, Dnmt3a, and Dnmt3b (A), and Tet1, Tet2, and Tet3 (B) was determined by ChIP-qPCR in the promoters of the 5hmC- enriched genes shown in Figure 4. The values are the averages + SD determined for three independent DNA samples.

Article Snippet: Solubilized chromatin was incubated with mouse monoclonal IgG (Cat No. 12-371, Millipore), rabbit IgG (Cat No. 12-370, Millipore), anti-Dnmt1 mouse monoclonal Dnmt1 (clone: 60B1220.1, Cat No. IMG-261A, Imgenex), anti-Dnmt3a/3a2 [5], anti-Dnmt3b [2], anti-Tet1 (Cat. No. 09-872, Millipore), antiTet2 (Cat No. R1086-6b, Abiocode), or anti-Tet3 (Cat. No. 61395, Activemotif) antibodies at 4°C overnight.

Techniques: ChIP-qPCR

Journal: Journal of Virology

Article Title: Interplay between the Epigenetic Enzyme Lysine (K)-Specific Demethylase 2B and Epstein-Barr Virus Infection

doi: 10.1128/JVI.00273-19

Figure Lengend Snippet: Methylation-dependent silencing of KDM2B expression. (A) LCL were cultured for 96 h in the presence of DMSO (nontreated [NT]) or 5-aza-2′-deoxycytidine (Aza). Cells were then collected, and the KDM2B expression levels were analyzed by RT-qPCR. The histogram shows the average KDM2B mRNA levels measured in 3 independent experiments (*, P < 0.05). KDM2B mRNA levels in Aza-treated LCL were measured relative to its levels in DMSO-treated cells. (B) LCL were transfected with stabilized siRNA targeting DNMT1 (siDNMT1) in two independent experiments. At 4 days after transfection, cells were collected and analyzed for the protein levels of DNMT1 (top) and the mRNA levels of KDM2B (bottom). KDM2B mRNA levels in DNMT1-targeted siRNA-treated cells were measured relative to its levels in cells treated with scrambled (scr) siRNA. (C) RPMI cells infected with EBV or mock infected were fixed and processed for ChIP with a DNMT1 antibody or an IgG antibody as a negative control. The eluted DNA was analyzed by qPCR with primers flanking CpG15695155, CpG21423404, and CpG island 127 (primer sequences are described in Table 3) (*, P < 0.05; ns, not significant).

Article Snippet: The following antibodies were used: KDM2B (catalog number ab5199; Abcam), DNMT1 (catalog number MAB0079; Abnova), and IgG (Diagenode).

Techniques: Methylation, Expressing, Cell Culture, Quantitative RT-PCR, Transfection, Infection, Negative Control

Journal: Journal of Virology

Article Title: Interplay between the Epigenetic Enzyme Lysine (K)-Specific Demethylase 2B and Epstein-Barr Virus Infection

doi: 10.1128/JVI.00273-19

Figure Lengend Snippet: Primers used for qPCR and ChIP-qPCR

Article Snippet: The following antibodies were used: KDM2B (catalog number ab5199; Abcam), DNMT1 (catalog number MAB0079; Abnova), and IgG (Diagenode).

Techniques: Sequencing

Journal: Journal of Virology

Article Title: Interplay between the Epigenetic Enzyme Lysine (K)-Specific Demethylase 2B and Epstein-Barr Virus Infection

doi: 10.1128/JVI.00273-19

Figure Lengend Snippet: LMP1 mediates downregulation of KDM2B. (A) RPMI cells were stably transduced with pLXSN or with pLXSN-LMP1 (LMP1) in four independent transduction experiments. Cells were then collected, and the expression levels of LMP1 (left) and KDM2B (right) were analyzed by RT-qPCR (****, P < 0.0001). (B) RPMI-pLXSN or RPMI-LMP1 cells were cultured in the presence of Aza (+) or DMSO (−) for 48 h, and the KDM2B mRNA expression level was analyzed by RT-qPCR. The histogram shows the average from 2 independent experiments (*, P < 0.05; ns, not significant). (C and D) Louckes cells were stably transduced with pLXSN, pLXSN-LMP1 (LMP1), or pLXSN-LMP1 mutants (3AAA, 378, and 3AAA/378). Cells were collected and processed for RNA and protein analysis. mRNA expression and protein levels of LMP1 were detected by RT-PCR (C, top) and immunoblotting (C, bottom). KDM2B mRNA and protein levels were also shown by RT-qPCR and immunoblotting, respectively (D, left and right). In panel D, the difference between KDM2B mRNA and protein levels in Louckes cells with pLXSN and in Louckes cells stably expressing wild-type LMP1 was significant (*, P < 0.05; **, P < 0.01). The levels of KDM2B mRNA in cells expressing wild-type (WT) LMP1 and LMP1 mutants were measured relative to its level in cells expressing pLXSN. (E) RPMI-LMP1 cells were treated for 2 h with BAY11-7082 (Bay11) (10 μM) or with JNK inhibitor II (JNKII) (10 μM) in three independent experiments. KDM2B mRNA levels were analyzed by RT-qPCR (*, P < 0.05). (F) RPMI pLXSN and RPMI-LMP1 cells, the latter of which were untreated or treated with BAY11-7082 (10 μM) for 2 h, were fixed to perform ChIP with DNMT1 or IgG antibodies. The eluted DNA was analyzed by qPCR with primers designed to surround CpG21423404. The histogram shows the average percentage of recruitment of DNMT1 in 4 independent experiments (*, P < 0.05).

Article Snippet: The following antibodies were used: KDM2B (catalog number ab5199; Abcam), DNMT1 (catalog number MAB0079; Abnova), and IgG (Diagenode).

Techniques: Stable Transfection, Transduction, Expressing, Quantitative RT-PCR, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: International Journal of Oncology

Article Title: TIP60 governs the auto-ubiquitination of UHRF1 through USP7 dissociation from the UHRF1/USP7 complex

doi: 10.3892/ijo.2021.5269

Figure Lengend Snippet: TIP60 interferes with UHRF1-USP7 association and their expression levels. HeLa cells were transfected with either TIP60-eGFP WT or TIP60ΔMYST-eGFP mutant. Western blot and immunoprecipitated samples were resolved by SDS-PAGE and immunoblotted with anti-UHRF1, anti-USP7 and anti-DNMT1 antibodies. (A) Effect of TIP60 on UHRF1, USP7 and DNMT1 levels with or without MG-132 treatment, respectively. (B and C) Quantification of the effect of TIP60 on UHRF1, USP7 and DNMT1 levels with or without MG-132 treatment, respectively. Values are the mean ± SEM from at least three independent experiments which were analyzed statistically by one-way ANOVA with Tukey's post hoc test ( * P<0.05; *** P<0.001 vs. control group). (D) Anti-UHRF1 antibody was used to co-immunoprecipitate UHRF1 and its partner USP7. (E) In a reciprocal experiment, anti-USP7 antibody was used to co-immunoprecipitate USP7 and UHRF1. Inputs and IP gels were processed in parallel under similar conditions. UHRF1, ubiquitin-like, containing PHD and RING finger domains 1; TIP60, Tat interactive protein, 60 kDa; USP7, ubiquitin-specific-processing protease 7; DNMT1, DNA methyltransferase 1.

Article Snippet: Other antibodies used included rabbit polyclonal anti-HAUSP/USP7 (1:5,000; cat. no. ab4080, Abcam), mouse monoclonal anti-DNMT1 (1:5,000; cat. no. PTG-MAB0079, ProteoGenix), mouse monoclonal anti-ubiquitin (1:500; cat. no. 05-944, Sigma-Aldrich; Merck KGaA), mouse monoclonal eGFP (1:1,000; cat. no. 66,002-1-Ig, Proteintech Group, Inc.; and cat. no. A-11120, Thermo Fisher Scientific, Inc.), mouse monoclonal anti-GAPDH (1:5,000; cat. no. MAB374, Merck KGaA), mouse monoclonal anti-GFP (1:1,000; cat. no. 66002-1-Ig, Proteintech Group, Inc.), mouse monoclonal anti-p73 (1:500; cat. no. 558785, BD Biosciences), rabbit polyclonal anti-caspase-3 (1:1,000; cat. no. 9661, Cell Signaling Technology, Inc.), mouse monoclonal anti-BCL2 (1:1,000; cat. no. 05-826, Merck KGaA), mouse monoclonal anti-poly(ADP-ribose) polymerase (PARP; 1:1,000; cat. no. 51-6639GR, BD Biosciences) and rabbit polyclonal anti-BAX (1:1,000; cat. no. AB2930, Merck KGaA).

Techniques: Expressing, Transfection, Mutagenesis, Western Blot, Immunoprecipitation, SDS Page