Journal: Aging cell

Article Title: DNMT3a Deficiency Contributes to Anesthesia/Surgery-Induced Synaptic Dysfunction and Cognitive Impairment in Aged Mice.

doi: 10.1111/acel.14458

Figure Lengend Snippet: FIGURE 2 | Anesthesia/surgery decreased the expression of DNMT3a mRNA and protein in hippocampal neurons. (A) Heatmap of genes associ- ated with DNA methylation. (B) Relative expression of genes associated with DNA methylation from hippocampal (n = 5). (C) Protein blotting bands for DNMT3a in hippocampus tissues and quantification of relative protein expression (n = 6). Representative images of DNMT3a co-stained with different cell types. (D) Neurons (NeuN) or (E) astrocytes (GFAP) or (F) microglia (Iba1) in the hippocampal CA1 region. Quantification of DNMT3a intensity in different types of cells: (G) microglia or (H) astrocytes or (I) neurons (n = 9). All values are presented as mean ± SEM ( **p < 0.01, and ***p < 0.001, unpaired t test for B, G, H, I and one-way ANOVA with Dunnett's test for C).

Article Snippet: Primary antibodies 5- mC (28692, CST), 5- hmC (ab214728, Abcam), DNMT3a (20954, Proteintech), NeuN (MAB377, Milipore), GFAP (ab4674, Abcam), Iba1 (ab5076, Abcam), Flag (8146, CST), LRG1 (SC390920, Santa Cruz) were applied overnight at 4°C and secondary antibodies Donkey Anti- Rabbit 594 (ab150064, Abcam), Donkey Anti- Mouse Cy5 (AB_2340819, Jackson ImmunoResearch), Donkey Anti- Chicken 488 (ab63507, Abcam), Donkey AntiGoat 488 (ab150129, Abcam) for 2 h at room temperature.

Techniques: Expressing, DNA Methylation Assay, Staining

Journal: Aging cell

Article Title: DNMT3a Deficiency Contributes to Anesthesia/Surgery-Induced Synaptic Dysfunction and Cognitive Impairment in Aged Mice.

doi: 10.1111/acel.14458

Figure Lengend Snippet: FIGURE 3 | DNMT3a knockdown in the hippocampus leads to decreased DNA methylation levels, synaptic disorder, and memory deficiency. (A) Schematic of the experimental paradigm. (B) Representative fluorescence image of the AAV-infected slice. (C) Protein blotting bands for DNMT3a. (D) Quantification of relative protein expression. (E) Relative gene expression in hippocampus (n = 6). (F, G) Representative field and quantification of 5-mC expression in the CA1 (n = 9). (H) Global DNA methylation level in the hippocampus (n = 6). (I) Normalized the fEPSP slope and amplitude at hippocampal. Quantitative analysis of fEPSP slope and amplitude at last 20 min (n = 5). (J) Golgi staining images showing the dendritic trees in hippocampus. The Sholl analysis was performed to evaluate the dendritic complexity (n = 5). The average speed (K), time spent in the central area (L), and representative movement track (M) in OFT (n = 12). The escape latency over the training session (N). The percentage of times spent in the target quadrant (O) and representative movement tracks (P) (n = 12). (Q) The percentage of freezing times (n = 12). All values are presented as mean ± SEM (*p < 0.05, **p < 0.01, unpaired t-test for D, E, G, H, I, K, L, O, Q and two-way ANOVA with Bonferroni post hoc test for J).

Article Snippet: Primary antibodies 5- mC (28692, CST), 5- hmC (ab214728, Abcam), DNMT3a (20954, Proteintech), NeuN (MAB377, Milipore), GFAP (ab4674, Abcam), Iba1 (ab5076, Abcam), Flag (8146, CST), LRG1 (SC390920, Santa Cruz) were applied overnight at 4°C and secondary antibodies Donkey Anti- Rabbit 594 (ab150064, Abcam), Donkey Anti- Mouse Cy5 (AB_2340819, Jackson ImmunoResearch), Donkey Anti- Chicken 488 (ab63507, Abcam), Donkey AntiGoat 488 (ab150129, Abcam) for 2 h at room temperature.

Techniques: Knockdown, DNA Methylation Assay, Fluorescence, Infection, Expressing, Gene Expression, Staining

Journal: International Journal of Molecular Sciences

Article Title: Maternal Methyl Donors Supplementation during Lactation Prevents the Hyperhomocysteinemia Induced by a High-Fat-Sucrose Intake by Dams

doi: 10.3390/ijms141224422

Figure Lengend Snippet: Correlation analysis between plasma homocysteine levels (μmol/L) and hepatic Dnmt3a mRNA levels (AU). Male rats are represented by squares and females by crosses.

Article Snippet: Quantitative real-time PCR ( n = 6 animals from 5–6 L per maternal dietary group) was performed by triplicate using ABI PRISM 7900 HT Fast real-time PCR system (Applied Biosystems, Austin, TX, USA) and Taqman primers (Applied Biosystems, Austin, TX, USA) for Dnmt1 (Rn00709664_m1*), Dnmt3a (Rn01027162_g1*) and Dnmt3b (Rn01536419_m1).

Techniques: Clinical Proteomics

Journal: Cancer Research and Treatment : Official Journal of Korean Cancer Association

Article Title: DNA Methyltransferase Gene Polymorphisms for Prediction of Radiation-Induced Skin Fibrosis after Treatment of Breast Cancer: A Multifactorial Genetic Approach

doi: 10.4143/crt.2016.256

Figure Lengend Snippet: Association analysis between polymorphisms of DNA methyltransferase genes and risk of grade ≥ 2 radiation-induced fibrosis (LENT-SOMA scale) in breast cancer patients

Article Snippet: Determination of SNPs was conducted on genomic DNA by real-time polymerase chain reaction (PCR) using the following TaqMan Pre-Designed SNP Genotyping assays (Applied Biosystems, Milan, Italy): C_27838930_10 ( DNMT1 rs2228611); C_8722920_10 ( DNMT3A rs1550117); C_7863728_10 ( DNMT3A rs7581217); C_16013055_10 ( DNMT3B rs2424908); and C_16269889_10 ( XRCC1 rs2682585).

Techniques: Significance Assay

Journal: PLoS Genetics

Article Title: Dnmt3a Is a Haploinsufficient Tumor Suppressor in CD8+ Peripheral T Cell Lymphoma

doi: 10.1371/journal.pgen.1006334

Figure Lengend Snippet: (A) Genetic setting used to conditionally delete Dnmt3a. The tetracycline activator protein (tTA) is expressed in ~40% of all hematopoietic cells (including stem cells). tTA promotes expression of the Teto-Cre transgene. Expression of Cre results in the excision of the stop cassette located upstream of the Rosa26LOXP EGFP reporter locus and deletion of Dnmt3a within the same subpopulation of cells. Thus, inclusion of the EGFP transgene allows for monitoring cells expressing tTA, and Cre, as well as to identify cells deleted for Dnmt3a. (B) PCR-based genotyping of the Dnmt3a locus using DNA isolated from FACS-sorted EGFP-positive (+) and EGFP-negative (−) populations on cells obtained from the bone marrow (BM), spleen (SP) and thymus (TH) of 6-week-old Dnmt3a −/− mice. PCR reactions were performed on samples in the following order, BM: (1) -LSK, (2) +LSK, SP: (1) -B2, (2) +B2, (3) -B1, (4) +B1, (5) -myeloid, (6) +myeloid, (7) -erythroid, (8) +erythroid, TH: (1) -DP, (2) +DP, (3) -CD4, (4) +CD4, (5) -CD8, (6) +CD8. Immunophenotypes of sorted populations are as follows: LSK (Lineage-, Sca-1+, ckit+), B2 (B220+, CD5-), B1 (B220+, CD5+), Myeloid (CD11b+), erythroid (Ter119+), DP (CD4+, CD8+), CD4 (CD4+, CD8-), CD8 (CD4-, CD8+). Fragments from floxed (F), wild-type (WT) and knockout (KO) alleles are shown. DNA from conventional Dnmt3a heterozygous (F/- and +/-) mice were used as controls. (C) Kaplan Meyer survival curve for Dnmt3a Δ/Δ mice. Moribund mice were classified at the time of terminal harvest with CLL (red), PTCL (yellow), or mixed CLL/PTCL (green) based on the presence of B-1a or CD8+ T cells tumors, as determined by FACS. Dnmt3a +/+ wild-type (+/+) controls are shown in blue. (D) A terminally ill Dnmt3a Δ/Δ mouse with PTCL. Generalized lymphadenopathy is denoted by arrows. (E) H&E stained sections of Dnmt3a +/+ (normal) and Dnmt3a Δ/Δ (PTCL) spleens (40X and 200X). (F) CD8 and CD4 expression in cells isolated from the lymph nodes (LN) and spleens (SP) of Dnmt3a +/+ control (+/+) and Dnmt3a Δ/Δ PTCL (Δ/Δ) mice as determined by FACS. Percentages of cells in each quadrant are shown in the top right in red. (G) CD3, CD8, TCR-β and TCR-γδ expression in cell isolated from the spleens (SP) of Dnmt3a +/+ control (+/+) and Dnmt3a Δ/Δ PTCL (Δ/Δ) mice, as determined by FACS. (H) Immunoblot showing Dnmt3a protein levels in Dnmt3a +/+ normal (N) controls (+/+) and Dnmt3a Δ/Δ PTCL (Δ/Δ) lymph node samples. γ-tubulin is shown as a loading control. (I) Time to tumor for primary (1), secondary (2) and tertiary (3) sub-lethally irradiated FVB-recipient mice serially transplanted with CD8+ PTCL tumors isolated from the lymph nodes of Dnmt3a Δ/Δ terminally sick mice. Data are presented as average time to tumor development. Three PTCL lines are shown.

Article Snippet: Western blots were performed as previously described [ ] with use of the following antibodies: Dnmt3a (H-295, Santa Cruz), γ-Tubulin (H-183, Santa Cruz), p53 (SC-6243, Santa Cruz), HDAC1 (ab7028, Abcam), and HSC-70 (SC-7298, Santa Cruz).

Techniques: Expressing, Isolation, Knock-Out, Staining, Control, Western Blot, Irradiation

Journal: PLoS Genetics

Article Title: Dnmt3a Is a Haploinsufficient Tumor Suppressor in CD8+ Peripheral T Cell Lymphoma

doi: 10.1371/journal.pgen.1006334

Figure Lengend Snippet: (A) Percentage of conventional heterozygous Dnmt3a +/- mice developing PTCL (yellow), MPD (green), CLL (red), or no disease (blue) at time of harvest, as determined FACS. (N = 30). (B) FACS analysis of CD8 and CD4 expression in cells isolated from the spleens (SP) and lymph nodes (LN) of Dnmt3a +/+ control (+/+) and Dnmt3a +/- PTCL (+/-) mice. Percentages of cells in each quadrant are shown in the top right in red. (C) H&E stained sections of Dnmt3a +/+ (normal) and Dnmt3a +/- (PTCL) spleens (40X and 200X). (D) The time to tumor development for primary (1), secondary (2) and tertiary (3) sub-lethally irradiated FVB-recipient mice serially transplanted with CD8+ PTCL tumors isolated from the lymph nodes of a Dnmt3a +/- terminally sick mice. Data are presented as average time to tumor development. Three PTCL lines are shown. (E) Immunoblot showing Dnmt3a protein levels in Dnmt3a +/+ controls (+/+) and Dnmt3a +/- PTCL (+/-) samples. Dnmt3a Δ/Δ PTCL (Δ/Δ) is shown as a negative control. γ-tubulin is shown as a loading control. (F) Representative flow diagram showing CD8 and TCR-Vβ 8.1/8.2 expression in Dnmt3a +/+ controls (+/+) and Dnmt3a +/- PTCL LN samples.

Article Snippet: Western blots were performed as previously described [ ] with use of the following antibodies: Dnmt3a (H-295, Santa Cruz), γ-Tubulin (H-183, Santa Cruz), p53 (SC-6243, Santa Cruz), HDAC1 (ab7028, Abcam), and HSC-70 (SC-7298, Santa Cruz).

Techniques: Expressing, Isolation, Control, Staining, Irradiation, Western Blot, Negative Control

Journal: PLoS Genetics

Article Title: Dnmt3a Is a Haploinsufficient Tumor Suppressor in CD8+ Peripheral T Cell Lymphoma

doi: 10.1371/journal.pgen.1006334

Figure Lengend Snippet: (A) Percentage of differentially methylated CpGs (DMCS) in Dnmt3a Δ/Δ PTCL relative to wild-type CD8+ cells. Differentially methylated CpGs (DMCS) are defined as either hypo- (blue) or hypermethylated (orange) by a ≥30% change in percent CpG methylation in tumor samples compared to wild-type control samples. CpGs not meeting these criteria are shown in gray (unchanged). (B) Genomic location of hypomethylated (left) and hypermethylated (right) DMCS in Dnmt3a Δ/Δ PTCL, as compared to wild-type CD8+ cells. DMCS were annotated to long gene promoters (-1500 to +500bp relative to TSS), gene bodies, or intergenic regions. (C) Heat map comparing the methylation status of 21,712 promoters in wild-type CD8+ and Dnmt3a Δ/Δ PTCL samples, as determined by WGBS. The averaged percent CpG methylation at core promoter regions (-300bp to +150bp relative to the TSS, left ) and at long promoter regions (-1500bp to +500bp relative to the TSS, right ) are displayed. (D) The number of genes with differentially methylated regions (DMRS) in their long promoters, core promoters, gene bodies and predicted enhancers in Dnmt3a Δ/Δ PTCL relative to CD8+ control. (E) The number of hypo- and hypermethylated CpGs present in LINE, LTR, SINE, and DNA repeat elements in Dnmt3a Δ/Δ PTCL, as compared to wild-type CD8+ cells. (F) The number of AML1, NF-κB, and OCT1 transcription factor motifs present in hypomethylated promoters in Dnmt3a Δ/Δ PTCL, as compared to wild-type CD8+ cells (yellow). The average motif count of 12 randomly generated control promoter sets is shown in red, with error bars denoting standard deviation. (*) denotes p<0.05 by a Wilcoxon rank test. (G) The number of AP-2rep, SOX5, and myogenin transcription factor motifs present in hypermethylated promoters in Dnmt3a Δ/Δ PTCL, as compared to wild-type CD8+ cells (blue). The average motif count of 12 randomly generated control promoter sets is shown in red, with error bars denoting standard deviation. (*) denotes p<0.05 by a Wilcoxon rank test.

Article Snippet: Western blots were performed as previously described [ ] with use of the following antibodies: Dnmt3a (H-295, Santa Cruz), γ-Tubulin (H-183, Santa Cruz), p53 (SC-6243, Santa Cruz), HDAC1 (ab7028, Abcam), and HSC-70 (SC-7298, Santa Cruz).

Techniques: Methylation, CpG Methylation Assay, Control, Generated, Standard Deviation

Journal: PLoS Genetics

Article Title: Dnmt3a Is a Haploinsufficient Tumor Suppressor in CD8+ Peripheral T Cell Lymphoma

doi: 10.1371/journal.pgen.1006334

Figure Lengend Snippet: Circos plot of DMRS annotated to promoters and gene bodies in Dnmt3a Δ/Δ PTCL relative to wild-type CD8+ cells. DMRs aligning to promoter (outer circle) and gene body (inner circles) are displayed in relation to its chromosomal position in the mouse genome. Hypomethylated DMRS are indicated by yellow lines and hypermethylated DMRS are indicated by blue lines.

Article Snippet: Western blots were performed as previously described [ ] with use of the following antibodies: Dnmt3a (H-295, Santa Cruz), γ-Tubulin (H-183, Santa Cruz), p53 (SC-6243, Santa Cruz), HDAC1 (ab7028, Abcam), and HSC-70 (SC-7298, Santa Cruz).

Techniques:

Journal: PLoS Genetics

Article Title: Dnmt3a Is a Haploinsufficient Tumor Suppressor in CD8+ Peripheral T Cell Lymphoma

doi: 10.1371/journal.pgen.1006334

Figure Lengend Snippet: COBRA analysis of promoter methylation for Coro2a , Cxcr5 , Ikzf3 , IL2Rβ , Jdp2 , Lpar5 , Oas3 , Ppil1 , Pvt1 , Racgap1 and Wnt8a in wild-type CD8+, Dnmt3a Δ/Δ pre-tumor CD8+, Dnmt3a Δ/Δ PTCL, Dnmt3a +/- pre-tumor CD8+, and Dnmt3a +/- PTCL samples. PCR fragments amplified from bisulfite-treated genomic DNA were digested with the restriction enzymes BstU I, Taq 1 or Tai I. Undigested (U) and digested (D) fragments correspond to unmethylated and methylated DNA, respectively. Control PCR fragments generated from fully methylated mouse genomic DNA that is undigested (M) or digested (CpG) are shown.

Article Snippet: Western blots were performed as previously described [ ] with use of the following antibodies: Dnmt3a (H-295, Santa Cruz), γ-Tubulin (H-183, Santa Cruz), p53 (SC-6243, Santa Cruz), HDAC1 (ab7028, Abcam), and HSC-70 (SC-7298, Santa Cruz).

Techniques: Combined Bisulfite Restriction Analysis Assay, Methylation, Amplification, Control, Generated

Journal: PLoS Genetics

Article Title: Dnmt3a Is a Haploinsufficient Tumor Suppressor in CD8+ Peripheral T Cell Lymphoma

doi: 10.1371/journal.pgen.1006334

Figure Lengend Snippet: (A) Heat map of RNA-seq global expression data displaying differentially expressed genes in Dnmt3a +/- and Dnmt3a Δ/Δ PTCL (≥2 fold change and a p-value < 0.05) relative to wild-type CD8+ cells, 737 genes were overexpressed and 697 genes were underexpressed in both Dnmt3a +/- and Dnmt3a Δ/Δ PTCL. 650 overexpressed and 549 underexpressed genes were specific to Dnmt3a +/- PTCL, whereas 329 overexpressed and 185 underexpressed genes were only observed in Dnmt3a Δ/Δ PTCL. A color bar displays fold change in gene expression with overexpressed shown in red and underexpressed in green. (B) Venn diagram showing overlap in over- and underexpressed genes between Dnmt3a +/- PTCL (purple) and Dnmt3a Δ/Δ PTCL (gray). (C) (left) Venn diagram showing overlap between the number of differentially expressed genes (red = overexpression; green = underexpression) and the number of differentially methylated gene promoters (yellow = hypomethylation; blue = hypermethylation) in Dnmt3a Δ/Δ PTCL relative to wild-type CD8+ controls. (right) Overlap between differentially expressed genes and differentially methylated enhancers regions. (D) List of genes hypomethylated and overexpressed in Dnmt3a Δ/Δ PTCL (HOT genes). Promoter methylation for wild-type CD8+ cells and Dnmt3a Δ/Δ PTCL is shown in blue and yellow, respectively. Corresponding changes in gene expression for Dnmt3a Δ/Δ PTCL relative to control CD8+ samples are shown in red.

Article Snippet: Western blots were performed as previously described [ ] with use of the following antibodies: Dnmt3a (H-295, Santa Cruz), γ-Tubulin (H-183, Santa Cruz), p53 (SC-6243, Santa Cruz), HDAC1 (ab7028, Abcam), and HSC-70 (SC-7298, Santa Cruz).

Techniques: RNA Sequencing, Expressing, Gene Expression, Over Expression, Methylation, Control

Journal: PLoS Genetics

Article Title: Dnmt3a Is a Haploinsufficient Tumor Suppressor in CD8+ Peripheral T Cell Lymphoma

doi: 10.1371/journal.pgen.1006334

Figure Lengend Snippet: (A) Heat maps derived from global expression profiling for genes differentially expressed in both human PTCL (PTCL) relative to normal tonsil T cells (T) and in Dnmt3a +/- PTCL (+/-) and/or Dnmt3a Δ/Δ PTCL (Δ/Δ) relative to normal Dnmt3a +/+ CD8+ cells. 182 genes were commonly overexpressed in human PTCL, Dnmt3a +/- PTCL, and Dnmt3a Δ/Δ PTCL, while 210 genes were commonly underexpressed in all three tumor types. 134 overexpressed and 205 underexpressed genes were specific to human PTCL and Dnmt3a +/- PTCL, whereas 70 overexpressed and 29 underexpressed genes were only observed in human PTCL and Dnmt3a Δ/Δ PTCL. For microarray data (human samples), genes with a fold change >1.5 and a P-value <0.05 were considered significant. For RNA-seq data (mouse samples), genes with a fold change >2 and a p-value <0.05 were considered significant. A color bar displays fold change in gene expression with overexpressed shown in red and underexpressed in green. (B) Venn diagrams showing overlaps in gene expression between human PTCL (black) and mouse Dnmt3a +/- PTCL (blue) and Dnmt3a Δ/Δ PTCL (green), as determined in panel A of the figure.

Article Snippet: Western blots were performed as previously described [ ] with use of the following antibodies: Dnmt3a (H-295, Santa Cruz), γ-Tubulin (H-183, Santa Cruz), p53 (SC-6243, Santa Cruz), HDAC1 (ab7028, Abcam), and HSC-70 (SC-7298, Santa Cruz).

Techniques: Derivative Assay, Expressing, Microarray, RNA Sequencing, Gene Expression

Journal: PLoS Genetics

Article Title: Dnmt3a Is a Haploinsufficient Tumor Suppressor in CD8+ Peripheral T Cell Lymphoma

doi: 10.1371/journal.pgen.1006334

Figure Lengend Snippet: (A) COBRA analysis of mouse Jdp2 promoter methylation in three independent Dnmt3a Δ/Δ PTCL samples. Undigested (U) and digested (D) fragments correspond to unmethylated and methylated DNA, respectively. Control PCR fragments generated from fully methylated mouse genomic DNA that is undigested (M) or digested (CpG) are shown. (B) Normalized gene expression of Jdp2 in mouse CD8+ control, Dnmt3a +/- PTCL, and Dnmt3a Δ/Δ PTCL samples by qRT-PCR. Data presented are the average of two independent experiments. Error bars show standard deviation and an asterisk (*) denotes a p<0.05 (student t-test). (C) Bisulfite sequencing of the JDP2 promoter in normal human CD3+ T cells and in two independent human PTCL samples. Circles represent individual CpGs within the promoter. Black and white areas denote the relative portion of methylated and un-methylated sequence reads at a CpG, respectively. (D) Normalized gene expression of JDP2 in normal human CD3+ T cells and human PTCL samples, by qRT-PCR. Data presented are the average of two independent experiments. Error bars show standard deviation and an asterisk (*) denotes a p<0.05 (student t-test).

Article Snippet: Western blots were performed as previously described [ ] with use of the following antibodies: Dnmt3a (H-295, Santa Cruz), γ-Tubulin (H-183, Santa Cruz), p53 (SC-6243, Santa Cruz), HDAC1 (ab7028, Abcam), and HSC-70 (SC-7298, Santa Cruz).

Techniques: Combined Bisulfite Restriction Analysis Assay, Methylation, Control, Generated, Gene Expression, Quantitative RT-PCR, Standard Deviation, Methylation Sequencing, Sequencing

Journal: PLoS Genetics

Article Title: Dnmt3a Is a Haploinsufficient Tumor Suppressor in CD8+ Peripheral T Cell Lymphoma

doi: 10.1371/journal.pgen.1006334

Figure Lengend Snippet: (A) Immunoblot showing p53 protein levels in Dnmt3a +/+ control spleen (SP), Dnmt3a +/- PTCL (+/-) Dnmt3a Δ/Δ PTCL (Δ/Δ) samples. HDAC1 is shown as a loading control. (B) Heatmap showing fold change in gene expression data derived from RNA-seq of 181 p53 pathway genes identified through GSEA for Dnmt3a +/- PTCL and Dnmt3a Δ/Δ PTCL relative to CD8+ controls. (C) RNA-seq expression profiles on Dnmt3a +/- PTCL, and Dnmt3a Δ/Δ PTCL were subjected to GSEA to identify enriched signatures. Each group was run as a pairwise comparison to normal CD8+ cells. In both tumor groups, the p53 pathway was identified as being significantly downregulated relative to controls. Normalized enrichment scores (NES), false discovery rate (FDR) and P-values are shown for each analysis. Black bars indicate individual genes within the pathway. Red indicates genes with high expression and blue indicates low expression in tumors relative to controls. (D) Immunoblot showing p53 protein levels in Dnmt3a +/+ (+/+) and Dnmt3a +/- (+/-) cells isolated from the spleen (SP), thymus (TH), and lymph node (LN) of 9 month old mice with no signs of lymphoma development. HSC-70 is shown as a loading control. (E) Immunoblot showing p53 protein levels in Dnmt3a +/+ (+/+) and Dnmt3a +/- (+/-) cells isolated from the spleen (SP) and thymus (TH) of 6 week and 9 month old mice with no signs of lymphoma development. HSC-70 is shown as a loading control. (F) Relative percentage of EGFP positive cells as determined by FACS for Dnmt3a +/+ (+/+) and Dnmt3a -/- (-/-) lymphoma cell lines infected with either MSCV-IRES-EGFP (Vec) or MSCV-IRES-p53-EGFP (p53). EGFP measurements were taken at multiples time points and normalized by the percentage of EGFP positive cells observed 2 days post-transduction.

Article Snippet: Western blots were performed as previously described [ ] with use of the following antibodies: Dnmt3a (H-295, Santa Cruz), γ-Tubulin (H-183, Santa Cruz), p53 (SC-6243, Santa Cruz), HDAC1 (ab7028, Abcam), and HSC-70 (SC-7298, Santa Cruz).

Techniques: Western Blot, Control, Gene Expression, Derivative Assay, RNA Sequencing, Expressing, Comparison, Isolation, Infection, Transduction