Journal: Nucleic Acids Research

Article Title: Dissecting the behavior and function of MBD3 in DNA methylation homeostasis by single-molecule spectroscopy and microscopy

doi: 10.1093/nar/gkv098

Figure Lengend Snippet: Analysis of single-molecule brightness by PCH implicates a transition of MBD3 binding stoichiometry. ( A ) Photon counting histograms from nuclear MBD3-GFP in different cell phases are presented along with corresponding images. ( B ) Weighted average brightness of different GFP tagged constructs was obtained, with pure GFP as the brightness standard, TLR9-GFP as the dimer control and MBD1-GFP as the monomer control. ( C ) Dimer percentage for each protein was calculated based on the brightness of pure GFP in buffer. ( D ) Hypotonic medium disrupted the dimer formation of MBD3-GFP in the G1-phase nucleus. Scale bar: 10 μm.

Article Snippet: MBD3 siRNA, scrambled siRNA and transfection reagents for knockdown experiments were obtained from Santa Cruz Biotechnology.

Techniques: Binding Assay, Construct, Control

Journal: Nucleic Acids Research

Article Title: Dissecting the behavior and function of MBD3 in DNA methylation homeostasis by single-molecule spectroscopy and microscopy

doi: 10.1093/nar/gkv098

Figure Lengend Snippet: MBD3 transcription is concurrent with DNMTs during cell cycle progression. ( A ) Sequential cell synchronization was validated with flow cytometry followed by ( B ) quantitative RT-PCR for MBD3 and DNMTs. Relative quantity of each gene is normalized to the transcriptional level in the G1-phase and presented as the mean with standard deviation ( n = 4). Student's t -test was used to determine the statistical significance.

Article Snippet: MBD3 siRNA, scrambled siRNA and transfection reagents for knockdown experiments were obtained from Santa Cruz Biotechnology.

Techniques: Flow Cytometry, Quantitative RT-PCR, Standard Deviation

Journal: Nucleic Acids Research

Article Title: Dissecting the behavior and function of MBD3 in DNA methylation homeostasis by single-molecule spectroscopy and microscopy

doi: 10.1093/nar/gkv098

Figure Lengend Snippet: Restriction in the dynamics of nulcear MBD3 increases during cell cycle progression. ( A ) Illustration of FLCS data collection within different compartments (red cross for nucleus, white cross for cytoplasm) is shown. 2–3 measurement points were randomly chosen from each compartments. ( B ) Fitting autocorrelation curvers in FLCS: for cytoplamic MBD3-GFP (upper panel) a single-component 3D free diffusion model was used, while for nuclear MBD3-GFP (lower panel) an anomalous diffusion model was applied. ( C ) The MBD3-GFP diffusion times under different circumstances were obtained. Each group was summarized based on data from more than 15 cells (mean + standard error of the mean, n > 30). The ‘unbound’ measurements were mostly obtained from the free MBD3-GFP in cytoplasm.

Article Snippet: MBD3 siRNA, scrambled siRNA and transfection reagents for knockdown experiments were obtained from Santa Cruz Biotechnology.

Techniques: Diffusion-based Assay

Journal: Nucleic Acids Research

Article Title: Dissecting the behavior and function of MBD3 in DNA methylation homeostasis by single-molecule spectroscopy and microscopy

doi: 10.1093/nar/gkv098

Figure Lengend Snippet: The MBD3–MBD2–DMNT1 complex in DNA maintenance methylation was assessed by FLIM-FRET. ( A ) Labeled primary antibodies were applied to target the protein pairs (MBD3–DNMT1, MBD3–MBD2). ( B ) A shortened fluorescence lifetime due to FRET is reflected by the blue-shift in FLIM images (an in vitro antibody-mediated FLIM-FRET is illustrated). ( C ) FLIM images of the Alexa488 labeled onto anti-MBD3-IgG were obtained in different cell phases, with DNMT1 or MBD2 directly labeled with Alexa555. Scale bar: 10 μm. ( D ) The ensemble-based fluorescence lifetimes are presented with mean and stand deviation ( n = 10 images). FRET efficiencies were accordingly calculated (inset).

Article Snippet: MBD3 siRNA, scrambled siRNA and transfection reagents for knockdown experiments were obtained from Santa Cruz Biotechnology.

Techniques: Methylation, Labeling, Fluorescence, In Vitro

Journal: Nucleic Acids Research

Article Title: Dissecting the behavior and function of MBD3 in DNA methylation homeostasis by single-molecule spectroscopy and microscopy

doi: 10.1093/nar/gkv098

Figure Lengend Snippet: The dynamic characteristics of MBD3 in DNA methylation homeostasis. In the G1-phase, MBD3/NuRD adopts a ‘two-site sequential binding mode’ on its recognition CGIs and facilitates maintaining a hypomethylated state. k d1 and k d2 denote the two dissociation constants for the homodimeric binding. As the DNA starts to replicate, the newly synthesized hemi-methylated DNA intends to inherit the average methylation density from the parental template, conducted by DNMTs (mainly DNMT1). A proportion of MBD3 and MBD2 (a bona fide 5mC binding protein) would co-operate with DNMT1 herein to complement the accuracy of maintenance methylation. The demethylating potential of MBD3 and MBD2 provides a protective mechanism contributing to DNA methylation homeostasis in the S-G2 phases.

Article Snippet: MBD3 siRNA, scrambled siRNA and transfection reagents for knockdown experiments were obtained from Santa Cruz Biotechnology.

Techniques: DNA Methylation Assay, Binding Assay, Synthesized, Methylation

Journal: Nucleic Acids Research

Article Title: Dissecting the behavior and function of MBD3 in DNA methylation homeostasis by single-molecule spectroscopy and microscopy

doi: 10.1093/nar/gkv098

Figure Lengend Snippet: Disrupted DNA methylation homeostasis due to insufficient MBD3. ( A ) The siRNA knockdown effect was verified by quantitative RT-PCR and immunofluorescence. Upon MBD3 knockdown, ( B ) the global DNA methylation level was quantified with colorimetric immunoassay, ( C ) and the promoter CGIs methylation of 22 cell cycle related genes was quantified with PCR Array (presented as mean with standard deviation, n = 3). ( D ) Seventy-two hours after knockdown, the cell cycle distribution was analyzed by flow cytometry.

Article Snippet: MBD3 siRNA, scrambled siRNA and transfection reagents for knockdown experiments were obtained from Santa Cruz Biotechnology.

Techniques: DNA Methylation Assay, Knockdown, Quantitative RT-PCR, Immunofluorescence, Methylation, Standard Deviation, Flow Cytometry