Journal:

Article Title: Neither DNA hypomethylation nor changes in the kinetics of erythroid differentiation explain 5-azacytidine's ability to induce human fetal hemoglobin

doi: 10.1182/blood-2007-06-093948

Figure Lengend Snippet: siRNA knock-down of DNMT1 produces decreased γ-globin promoter methylation but no increase in γ-globin mRNA or fetal hemoglobin. siRNA for the DNMT1 gene was transiently transfected into cells on day +11 of in vitro erythroid differentiation. Controls for this experiment were cells from the same initial culture that were treated with a nonspecific siRNA (siCTRL) or underwent mock transfection without siRNA (CTRL). (A) The effect of siRNA treatment on DNMT1 mRNA levels during differentiation as determined by quantitative RT-PCR. (B) The effect of siRNA treatment on DNMT3a mRNA levels. (C) The effect of siRNA treatment on DNMT1 protein levels as determined by Western blotting. β-actin and DNMT3a serve as nonspecific controls. (D) The effect of siRNA treatment on global DNA methylation as determined by bisulfite conversion analysis of LINE elements. Note that for the day 15 control sample no error bars are shown. This is because all 5 sequences, which included a total of 42 individual CpGs, were 100% methylated so the standard deviation for this data point was 0. (E) The effect of siRNA treatment on γ-globin promoter DNA methylation. (F) The effect of siRNA treatment on γ-globin mRNA during differentiation. (G) The effect of siRNA treatment on hemoglobin production as assessed by HPLC analysis of cell lysates at the end of in vitro differentiation. CD34+ cells from a single normal donor were used for this experiment. Error bars represent 1 SD. P values were determined by t test.

Article Snippet: 17 DNMT1 siRNA and shRNA siRNA for DNMT1 or a fluorescein-labeled control siRNA (Santa Cruz Biotechnology, Santa Cruz, CA) were transfected into cells using HiPerFect reagent (Qiagen, Valencia, CA) following the manufacturer's suggested protocol for transfection of suspension cells on day +11 of the standard culture procedure.

Techniques: Knockdown, Methylation, Transfection, In Vitro, Quantitative RT-PCR, Western Blot, DNA Methylation Assay, Control, Standard Deviation

Journal:

Article Title: Neither DNA hypomethylation nor changes in the kinetics of erythroid differentiation explain 5-azacytidine's ability to induce human fetal hemoglobin

doi: 10.1182/blood-2007-06-093948

Figure Lengend Snippet: shRNA knock-down of DNMT1 produces decreased γ-globin promoter methylation but no increase in γ-globin mRNA or fetal hemoglobin. Lentiviral vector LL3.7 containing DNMT1 shRNA and GFP sequences was used to transduce differentiating cells on day +5. On day +7 cells expressing GFP were sorted and allowed to differentiate into erythroid cells. Controls for this experiment were cells from the same initial culture transduced with vectors carrying a mismatched version of the DNMT1 shRNA, an empty vector, or cells that were untreated. Effects of DNMT1 shRNA on (A) DNMT1 steady-state mRNA levels during in vitro differentiation, (B) DNMT1 protein levels as determined by Western blot, (C) γ-globin promoter DNA methylation, (D) LINE element DNA methylation, (E) γ-globin steady-state mRNA, and (F) Hb levels as determined by HPLC at the end of differentiation. CD34+ cells from a single healthy donor were used for this experiment. Error bars represent 1 SD. P values were determined by t test.

Article Snippet: 17 DNMT1 siRNA and shRNA siRNA for DNMT1 or a fluorescein-labeled control siRNA (Santa Cruz Biotechnology, Santa Cruz, CA) were transfected into cells using HiPerFect reagent (Qiagen, Valencia, CA) following the manufacturer's suggested protocol for transfection of suspension cells on day +11 of the standard culture procedure.

Techniques: shRNA, Knockdown, Methylation, Plasmid Preparation, Transduction, Expressing, In Vitro, Western Blot, DNA Methylation Assay

Journal: Cancer research

Article Title: DAXX Suppresses Tumor-Initiating Cells in Estrogen Receptor-positive Breast Cancer following endocrine therapy

doi: 10.1158/0008-5472.CAN-19-1110

Figure Lengend Snippet: A. MCF-7 and T47D cells were treated with 0 or 5nM E2 for 3 days. DNMT1 and DAXX protein levels were detected by Western blot analysis using Actin as a loading control. B-C. MCF-7 and T47D cells were transfected with a mock vector (EV) or the human DAXX cDNA (DAXX) for 2 days and then re-transfected with the non-specific (SCBi) or DNMT1-specific (DNMT1i) siRNA for an additional day. B. DAXX and DNMT1 proteins were detected by Western blotting. C. After 7 days, mammospheres were imaged, isolated, measured, and %MFE was calculated. Bar graphs show mean ± S.D. %MFE from three independent experiments. Statistical significance was calculated using two-way ANOVA with a Tukey post-hoc test for multiple comparisons. Symbols denote statistical significance between EV and DAXX, SCBi and DNMT1 under DAXX conditions, and 0nM and 5nM E2: #= P< 0.05, @= P< 0.001. D. MCF-7 cells were transfected with a non-specific (SCBi), DAXX-specific (DAXXi), or DNMT1-specific (DNMT1i) siRNA for 2 days. Cells treated with 0 or 5nM E2 for 1 day. Total DNA was isolated and subjected to bisulfite treatment. Bisulfite converted DNA was amplified using SOX2 promoter-specific primers or NOTCH4-CpG island-specific primers that anneal to bisulfite-treated DNA. The PCR product was purified and sent for DNA sequencing. CpG sites that were read as “T” were considered unmethylated and sites read as “C” were considered methylated. Bar graphs show mean (total number of methylated CpG sites)/ (total CpG sites read × 100) ± S.D. from five independent experiments. Statistical significance was calculated using two-way ANOVA with a Tukey post-hoc test for multiple comparisons. Symbol denotes statistical significance between 0nM and 5nM E2 and SCBi and DAXXi/DNMT1i groups: @= P< 0.001.

Article Snippet: DNMT1 siRNA was purchased from Origene (Catalogue # SR301244).

Techniques: Western Blot, Transfection, Plasmid Preparation, Isolation, Amplification, Purification, DNA Sequencing, Methylation

Journal: Cell Death & Disease

Article Title: Hypermethylation of mitochondrial DNA in vascular smooth muscle cells impairs cell contractility

doi: 10.1038/s41419-020-2240-7

Figure Lengend Snippet: Vascular SMCs were subjected to vehicle (CL) or PDGF-BB (20 ng/mL) treatments for 24 h, and a accumulations of DNMT1, -3A, and -3B in nuclear (Nucl.) and mitochondrial (Mito.) fractions were analyzed ( n = 3). Shown are representative blots. b Semi-quantification of DNMT1 and -3A in A. Expression was normalized to the respective internal reference proteins (VDAC1 or Histone 3) and expression in CL cells was set as 1. c , d Subcellular localization of DNMT1 was assessed by immunofluorescent staining followed by confocal microscopy ( c ) or structured illumination microscopy ( d ). Mito-tracker illustrates mitochondria. Arrowheads indicated co-localizations. Scale bar: 25 μm for c and 10 μm for d . e Co-localization of DNMT1 with mitochondria (mito-tracker) were quantified. Cells were randomly selected in microscopic fields from five biological replicates ( n = 45). f Fluorescence intensity profiles indicate the degree of overlap between DNMT1 and mitochondria. Scale bar: 25 μm. g Ratio of fluorescence intensity of DNMT1 in cytoplasm over nucleus was measured ( n = 40). h Schematic diagram of human mitochondrial DNA (h-mtDNA). Locations of primer sets for chromatin immunoprecipitation (ChIP) and methylation-specific PCR (MSP) were indicated. i DNMT1 bindings to D-loop regions in cells with CL treatment were analyzed by ChIP assay ( n = 5). j DNMT1 bindings to D-loop regions in CL- and PDGF-BB-treated cells were analyzed ( n = 5). k Methylation status of the D-loop region was measured by MSP ( n = 5). M: methylated; U: unmethylated. * P < 0.05 ** P < 0.01 *** P < 0.005 by student’s t -test ( b , i , j , and k ) and Mann–Whitney test ( e , g ), Error bars show ± SEM.

Article Snippet: Ad-shDNMT1 carrying short-hairpin RNA (shRNA) specifically targeting DNMT1 and the control adenovirus expressing GFP (Ad-GFP) were obtained from Vigene Biosciences.

Techniques: Expressing, Staining, Confocal Microscopy, Microscopy, Fluorescence, Chromatin Immunoprecipitation, Methylation, MANN-WHITNEY

Journal: Cell Death & Disease

Article Title: Hypermethylation of mitochondrial DNA in vascular smooth muscle cells impairs cell contractility

doi: 10.1038/s41419-020-2240-7

Figure Lengend Snippet: a Schematic diagram of the MTS-DNMT1 construct. MTS mitochondrial-targeting sequence, CMV cytomegalovirus promoter, ORF open-reading frame. b Representative immunofluorescent staining of mitochondria (Mito-tracker), no-MTS-DNMT1, and MTS-DNMT1 (DsRed2, Discosoma sp. red fluorescent protein 2). Cells were transfected either with no-MTS-DNMT1 (upper) or with MTS-DNMT1 (lower). Scale bar: 75 μm. c – j Vascular SMCs were transfected with empty vectors (pcDNA-3.1), no-MTS DNMT1, or MTS-DNMT1, and c The methylation status of D-loop region was measured by Methylation-specific PCR (MSP). ( n = 4). d The mRNA levels of mtDNA genes were detected by quantitative RT-PCR ( n = 7). e ROS production was detected by flow cytometry ( n = 4); f Mitochondrial membrane potential was measured by flow cytometry ( n = 3); g Intracellular ATP content was assessed by a luminometer ( n = 7); h MtDNA copy number was measured by quantitative RT-PCR assay ( n = 6); i Representative traces for oxygen consumption rates (OCR), which were assayed using a Seahorse XF24 flux analyzer, with sequential injections of mitochondrial effectors at time points indicated by the arrows ( n = 5); j The basal, ATP-linked, and maximal OCR in I were analyzed. ND NADH dehydrogenase, ATP ATP synthase, CO cytochrome c oxidase, Cytb Cytochrome b, ROS reactive oxygen species, M methylated, U unmethylated, FCCP carbonyl cyanide 4(trifluoromethoxy) phenylhydrazone * P < 0.05 ** P < 0.01 *** P < 0.005 by one-way ANOVA with Tukey’s post hoc analysis. Error bars show ± SEM.

Article Snippet: Ad-shDNMT1 carrying short-hairpin RNA (shRNA) specifically targeting DNMT1 and the control adenovirus expressing GFP (Ad-GFP) were obtained from Vigene Biosciences.

Techniques: Construct, Sequencing, Staining, Transfection, Methylation, Quantitative RT-PCR, Flow Cytometry, Membrane

Journal: Cell Death & Disease

Article Title: Hypermethylation of mitochondrial DNA in vascular smooth muscle cells impairs cell contractility

doi: 10.1038/s41419-020-2240-7

Figure Lengend Snippet: a Vascular SMCs were treated with control (CL) or oligomycin (2 or 5 μmol/L) for 12 h and intracellular ATP content was assessed by a luminometer ( n = 4). b – d Cells were embedded in collagen gel and were then treated with control (CL) or oligomycin (5 μmol/L) for 12 h in the absence ( b ) ( n = 4) or presence of doxycycline (1 μg/mL) ( c ) or roscovitine (10 μmol/L) pre-treatment (12 h) ( n = 5) ( d ). Cell contractions were determined by measuring the gel areas ( n = 4). e – g Cells were transfected with empty vectors, no-MTS DNMT1, or MTS-DNMT1 and were then subjected to gel contraction assay in the absence (E) ( n = 5) or presence of doxycycline (1 μg/mL) ( f ) or roscovitine (10 μmol/L) pre-treatment (12 h) ( n = 6). h Single-cell contractility of SMCs was measured by traction force microscopy (TFM). Upper: Representative cell force images. Lower: Quantification of traction force with respect to the indicated treatments ( n = 5). Scale bar: 25 μm. i Vascular SMCs were transfected with empty, no-MTS DNMT1, or MTS-DNMT1, and expressions of SMC contractile marker proteins were analyzed by western blot assay. Shown are representative blots ( n = 3). j Semi-quantification of proteins in i . * P < 0.05, ** P < 0.01, *** P < 0.005 by student’s t -test ( b – d ) and one-way ANOVA with Tukey’s post hoc analysis ( a , e , f – h , j ). Error bars show ± SEM.

Article Snippet: Ad-shDNMT1 carrying short-hairpin RNA (shRNA) specifically targeting DNMT1 and the control adenovirus expressing GFP (Ad-GFP) were obtained from Vigene Biosciences.

Techniques: Control, Transfection, Collagen Gel Contraction Assay, Microscopy, Marker, Western Blot

Journal: Cell Death & Disease

Article Title: Hypermethylation of mitochondrial DNA in vascular smooth muscle cells impairs cell contractility

doi: 10.1038/s41419-020-2240-7

Figure Lengend Snippet: a Schematic diagram for the process of mitochondrial transplantation. Mito. mitochondria. Mito-deleted cells were incubated with mitochondria prepared from the donor cells. Some donor cells were subjected to PDGF-BB or the vehicle treatments, others were transfected with MTS-DNMT1 plasmids or empty vectors. b Cells were treated with ethidium bromide (100 ng/mL) supplemented with sodium pyruvate (110 µg/mL), uridine (50 µg/mL) for 14 days to achieve mitochondrial deletion, and mtDNA copy numbers were detected by quantitative RT-PCR ( n = 4). c Live-cell confocal imaging for cells receiving mitochondria from the donors. The donors were transfected with MTS-DsRed2 plasmids (upper) or with empty vectors (middle). Images for cells undergoing no transplantation were also captured (lower). Scale bar: 25 μm. d , e Gel contraction assay ( n = 6) and traction force microscopy ( n = 4) in cells receiving mitochondria from PDGF-BB- (PDGF-mito) or CL-treated (CL-mito) cells. Scale bar: 25 μm. f , g Gel contraction assay ( n = 4) and traction force microscopy ( n = 4) in cells receiving mitochondria from MTS-DNMT1 (MTS-DNMT1-mito) or control vectors (Vector-mito) transfected cells. Scale bar: 25 μm. * P < 0.05, ** P < 0.01 by student’s t -test. Error bars show ± SEM.

Article Snippet: Ad-shDNMT1 carrying short-hairpin RNA (shRNA) specifically targeting DNMT1 and the control adenovirus expressing GFP (Ad-GFP) were obtained from Vigene Biosciences.

Techniques: Transplantation Assay, Incubation, Transfection, Quantitative RT-PCR, Imaging, Collagen Gel Contraction Assay, Microscopy, Control, Plasmid Preparation

Journal: Cell Death & Disease

Article Title: Hypermethylation of mitochondrial DNA in vascular smooth muscle cells impairs cell contractility

doi: 10.1038/s41419-020-2240-7

Figure Lengend Snippet: Mouse carotid arteries on both sides were isolated 1 or 4 weeks after ligation. a DNMT1 binding to D-loop region was analyzed by chromatin immunoprecipitation (ChIP) assay ( n = 3). b Transcriptional levels of 13 mtDNA genes were measured by quantitative RT-PCR ( n = 7). c – e Complex I, II, and IV-supported respiration was analyzed by Oroboros Oxygraph-2k, a high-resolution respiration analyzer ( n = 6). f ATP content was assessed by a luminometer n = 5. g , h Unligated or ligated carotid arteries were cut into ring segments of 3 mm in length and the ring segments were suspended in the myograph. KCl-simulated ( n = 6) and phenylephrine-simulated ( n = 4) vascular contraction were expressed as active tension (mN/mm). Phe phenylephrine. * P < 0.05, ** P < 0.01, *** P < 0.005 by student’s t -test ( a – e , f ) and one-way ANOVA with Tukey’s post hoc analysis ( h , i ). Error bars show ± SEM.

Article Snippet: Ad-shDNMT1 carrying short-hairpin RNA (shRNA) specifically targeting DNMT1 and the control adenovirus expressing GFP (Ad-GFP) were obtained from Vigene Biosciences.

Techniques: Isolation, Ligation, Binding Assay, Chromatin Immunoprecipitation, Quantitative RT-PCR