Journal: Cell Cycle

Article Title: RFTS-deleted DNMT1 enhances tumorigenicity with focal hypermethylation and global hypomethylation

doi: 10.4161/15384101.2014.950886

Figure Lengend Snippet: Deletion of RFTS enhances the oncogenic activity of DNMT1. (A) HBEC3 stable cell lines were established to express full-length and DNMT1 deletion forms near endogenous DNMT1 levels. The levels of DNMT1 were determined by RT-qPCR (left) and western blotting (right). Data were normalized to vector cells. (B) Adherent colony formation. (C) Soft-agar colony formation. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, indicates no significant difference in comparison to vector cells.

Article Snippet: Establishment of Stable Cell Lines To establish full length or deletion mutant human DNMT1 stable lines, a full-length DNMT1 cDNA clone (SC325419, Origene) was used as template to amplify full-length or deletion mutant DNMT1 fragments.

Techniques: Activity Assay, Stable Transfection, Quantitative RT-PCR, Western Blot, Plasmid Preparation

Journal: Cell Cycle

Article Title: RFTS-deleted DNMT1 enhances tumorigenicity with focal hypermethylation and global hypomethylation

doi: 10.4161/15384101.2014.950886

Figure Lengend Snippet: DNMT1-ΔRFTS promotes increased methylation and silencing of the DAPK and DUOX1 genes. (A) The methylation levels of DAPK (left) and DUOX1 (right) promoter-associated CpG islands were analyzed by qPCR. Methylated DNA was analyzed using the MethylMiner kit and amplified with specific primers. (B) Bisulfite sequencing results for DAPK (left) and DUOX1 (right) promoters. White squares represent unmethylated cytosines and black squares represent methylated cytosines in CpG sites. The percentage of methylated CpG dinucleotides from 8 independent clones is indicated. (C) DNMT1 chromatin occupancy was analyzed using DNMT1 ChIP and qPCR. (D) mRNA levels of DAPK (left) and DUOX1 (right) were analyzed by RT-qPCR and normalized to vector cells. *, p < 0.05; **, p < 0.01; ***, p < 0.001 in comparison to vector cells.

Article Snippet: Establishment of Stable Cell Lines To establish full length or deletion mutant human DNMT1 stable lines, a full-length DNMT1 cDNA clone (SC325419, Origene) was used as template to amplify full-length or deletion mutant DNMT1 fragments.

Techniques: Methylation, Amplification, Methylation Sequencing, Clone Assay, Quantitative RT-PCR, Plasmid Preparation

Journal: Cell Cycle

Article Title: RFTS-deleted DNMT1 enhances tumorigenicity with focal hypermethylation and global hypomethylation

doi: 10.4161/15384101.2014.950886

Figure Lengend Snippet: DNMT1-ΔRFTS decreases chromatin accessibility at DAPK and DUOX1 promoters. Cells were treated with or without DNA nuclease for 1 hr, prior to detection of promoter DNA by qPCR. The index of chromatin accessibility = 2 ((Ct DNase treated)-(Ct Untreated)). *, p < 0.05; ***, p < 0.001 in comparison to vector cells.

Article Snippet: Establishment of Stable Cell Lines To establish full length or deletion mutant human DNMT1 stable lines, a full-length DNMT1 cDNA clone (SC325419, Origene) was used as template to amplify full-length or deletion mutant DNMT1 fragments.

Techniques: Plasmid Preparation

Journal: Cell Cycle

Article Title: RFTS-deleted DNMT1 enhances tumorigenicity with focal hypermethylation and global hypomethylation

doi: 10.4161/15384101.2014.950886

Figure Lengend Snippet: 5-aza-dC treatment reactivates TSG expression and suppresses DNMT1-dependent transformation. (A) mRNA levels of DAPK (left) and DUOX1 (right) were analyzed by RT-qPCR after 100nM 5-aza-dC treatment for 5 d and normalized to vector cells treated with DMSO. (B) Soft-agar colony formation after 5-aza-dC treatment. ***, p < 0.001 in comparison to the DMSO treated control.

Article Snippet: Establishment of Stable Cell Lines To establish full length or deletion mutant human DNMT1 stable lines, a full-length DNMT1 cDNA clone (SC325419, Origene) was used as template to amplify full-length or deletion mutant DNMT1 fragments.

Techniques: Expressing, Transformation Assay, Quantitative RT-PCR, Plasmid Preparation

Journal: Cell Cycle

Article Title: RFTS-deleted DNMT1 enhances tumorigenicity with focal hypermethylation and global hypomethylation

doi: 10.4161/15384101.2014.950886

Figure Lengend Snippet: DNMT1-ΔRFTS expression enhances global DNMT1 methylation changes. (A) Genome-wide promoter DNA methylation profiles were obtained using the HELP assay. In volcano plots, the x-axis scores probe-specific methylation ratios and the y-axis scores p-values for the confidence of measurements. The plots allow visualization of methylation differences between vector and DNMT1 cells as well as the differences between vector and DNMT1-ΔRFTS cells. Probes sets that showed significant hyper- or hypomethylation (p < 0.05 for methylation changes (log2(HpaII/MspI)) > 2) are shown in cyan. All other probes are shown in red. (B) Heat map illustration of HpaII-enrichment fragments with methylation changes (log2(HapII/MspI)) > 2 between vector and DNMT1-ΔRFTS cells.

Article Snippet: Establishment of Stable Cell Lines To establish full length or deletion mutant human DNMT1 stable lines, a full-length DNMT1 cDNA clone (SC325419, Origene) was used as template to amplify full-length or deletion mutant DNMT1 fragments.

Techniques: Expressing, Methylation, Genome Wide, DNA Methylation Assay, HELP Assay, Plasmid Preparation

Journal: Cell Cycle

Article Title: RFTS-deleted DNMT1 enhances tumorigenicity with focal hypermethylation and global hypomethylation

doi: 10.4161/15384101.2014.950886

Figure Lengend Snippet: Genomic hypomethylation is found in DNMT1-ΔRFTS cells. (A) 5-methylcytosine (mC) content of the total cytosine pool was determined by HPLC. (B) Bisulfite sequencing of SAT2. White squares represent unmethylated CpGs, black squares represent methylated CpGs, and gray squares represent undetermined sites. Each row is an independent sequencing result. (C) Quantitation of SAT2 bisulfite sequencing. (D) DNMT1 chromatin occupancy was analyzed using DNMT1 ChIP and qPCR. (E) Expression of SAT2 non-coding RNA was analyzed by RT-qPCR and normalized to vector cells. *, p < 0.05; **, p < 0.01; ***, p < 0.001 in comparison to vector cells.

Article Snippet: Establishment of Stable Cell Lines To establish full length or deletion mutant human DNMT1 stable lines, a full-length DNMT1 cDNA clone (SC325419, Origene) was used as template to amplify full-length or deletion mutant DNMT1 fragments.

Techniques: Methylation Sequencing, Methylation, Sequencing, Quantitation Assay, Expressing, Quantitative RT-PCR, Plasmid Preparation

Journal: Cell Cycle

Article Title: RFTS-deleted DNMT1 enhances tumorigenicity with focal hypermethylation and global hypomethylation

doi: 10.4161/15384101.2014.950886

Figure Lengend Snippet: Dual roles for RFTS domain in DNMT1-dependent DNA methylation. (A) RFTS-targeted DNMT1 associated proteins (RAP) are proposed to relieve inhibition of DNMT1 for access to euchromatin. (B) The RFTS domain mediates association between DNMT1 and pericentromeric heterochromatin. (C) In cancer, overexpression of RAPs or mutation of RFTS is proposed to relieve DNMT1 inhibition, thereby increasing methylation and silencing of TSGs. However, because the RFTS domain is required for association with heterochromatic SAT2 sequences, DNMT1 with mutant RFTS may be less associated with such sequences, accounting for global hypomethylation.

Article Snippet: Establishment of Stable Cell Lines To establish full length or deletion mutant human DNMT1 stable lines, a full-length DNMT1 cDNA clone (SC325419, Origene) was used as template to amplify full-length or deletion mutant DNMT1 fragments.

Techniques: DNA Methylation Assay, Inhibition, Over Expression, Mutagenesis, Methylation

Journal: PLoS Pathogens

Article Title: HIV-1 Vpr-Induced Apoptosis Is Cell Cycle Dependent and Requires Bax but Not ANT

doi: 10.1371/journal.ppat.0020127

Figure Lengend Snippet: (A) pHR-VPR-G and pHR-VPR-R are bicistronic lentiviral vectors that encode HIV-1 NL4–3 vpr, an internal ribosome entry site, and either the gene for GFP or that for mRFP, respectively; pHR-VPR(R80A) was derived from pHR-VPR (both the mRFP and the GFP versions) by site-directed mutagenesis; DHIV3 is an envelope-truncated (see gray box) version of HIV-1 NL4–3 ; DHIV3-ΔVPR was derived from DHIV-3 by introducing a frameshift mutation in vpr . (B) SupT1 T lymphocytes were transduced by spin-infection in the presence of 10 μg/ml Polybrene with indicated vectors. Mock-infected cells were subjected to spin-infection in the presence of 10 μg/ml Polybrene without virus. Cells were collected at specified time points post-transduction, stained with propidium iodide, and analyzed for DNA content by flow cytometry to determine cell cycle profiles. The percentage of cells transduced with pHR vectors and DHIV3 viruses ranged between 70% to 80% and between 65% to 70%, respectively, as determined by mRFP or GFP expression (with pHR vectors) or intracellular p24 staining (with DHIV3 vectors). (C) Caspase activation was measured as an indication of apoptosis. SupT1 cells were infected with indicated vectors, harvested, and incubated with FITC-VAD-FMK. The percentage of caspase-active cells at each time point was measured by flow cytometry. (D) Infected SupT1 cells were lysed, and lysates were fractionated into mitochondrial (m) and cytoplasmic (c) fractions and then assayed by Western blot. Western blots were probed with antibodies specific to Smac to measure release from mitochondria and with anti-VDAC antibodies to measure mitochondrial contamination in the cytoplasmic fractions. As a positive control for apoptosis, SupT1 cells were treated with 0.8 μg/ml doxorubicin (dox) for 48 h. (E) Examples of flow cytometric analysis of caspase activation, corresponding to the 72-h time points from (C).

Article Snippet: For experiments in which viral transduction and measurement of cell cycle/apoptosis were the aims, we used the human T-cell line SupT1, which was maintained in RPMI 1640 (Cambrex BioScience), supplemented with 10% FCS and L-glutamine.

Techniques: Derivative Assay, Mutagenesis, Infection, Virus, Transduction, Staining, Flow Cytometry, Expressing, Activation Assay, Incubation, Western Blot, Positive Control

Journal: PLoS Pathogens

Article Title: HIV-1 Vpr-Induced Apoptosis Is Cell Cycle Dependent and Requires Bax but Not ANT

doi: 10.1371/journal.ppat.0020127

Figure Lengend Snippet: HeLa or Supt1 cells were transduced with pHR-VPR-G and at indicated time points, lysed, and analyzed by Western blot with a phospho-specific antibody that recognizes phosphorylation of H3 at serine-10. Nocodazole (250 ng/ml) and doxorubicin (1 μM) were used as positive and negative controls, respectively.

Article Snippet: For experiments in which viral transduction and measurement of cell cycle/apoptosis were the aims, we used the human T-cell line SupT1, which was maintained in RPMI 1640 (Cambrex BioScience), supplemented with 10% FCS and L-glutamine.

Techniques: Transduction, Western Blot, Phospho-proteomics

Journal: PLoS Pathogens

Article Title: HIV-1 Vpr-Induced Apoptosis Is Cell Cycle Dependent and Requires Bax but Not ANT

doi: 10.1371/journal.ppat.0020127

Figure Lengend Snippet: (A) SupT1 cells were infected with pHR-VPR-R or indicated mutants, at an MOI of 0.5. At 48 h postinfection, cells were stained with hypotonic PI to determine the cell cycle profiles. At 72 h postinfection, cells were incubated with FITC-VAD-FMK and analyzed by flow cytometry to determine the percentage of cells with active caspases. (B) Cells from above treatments were lysed at 48 h postinfection, and Western blot was performed to assay for phosphorylation of BRCA1 at Ser1423 by the ATR kinase. To establish the role of ATR in BRCA1 phosphorylation, parallel infections were treated with caffeine (2 mM). (C) Induction of apoptosis by Vpr-GFP and Vpr(R80A)-GFP fusion proteins. HPB-ALL cells were transfected with indicated constructs or mock-transfected and 48 after transfection, phosphatidylserine exposure was analyzed by flow cytometry using phycoerythrin-conjugated annexin V.

Article Snippet: For experiments in which viral transduction and measurement of cell cycle/apoptosis were the aims, we used the human T-cell line SupT1, which was maintained in RPMI 1640 (Cambrex BioScience), supplemented with 10% FCS and L-glutamine.

Techniques: Infection, Staining, Incubation, Flow Cytometry, Western Blot, Phospho-proteomics, Transfection, Construct