Journal: International Journal of Molecular Sciences

Article Title: Extracellular Vesicles from Human Teeth Stem Cells Trigger ATP Release and Promote Migration of Human Microglia through P2X4 Receptor/MFG-E8-Dependent Mechanisms

doi: 10.3390/ijms222010970

Figure Lengend Snippet: Characterization of extracellular vesicles (EVs) isolated from stem cells from the dental pulp of human exfoliated deciduous teeth (SHEDs). ( A ) Transmission electron microscopy of EVs isolated from SHEDs (120,000x magnification). ( B ) Concentration and particle size of EVs derived from SHEDs as analyzed by nanoparticle tracking analysis using a NanoSight LM10 instrument (Malvern Panalytical). Size distribution of the EVs was around 150 nm. ( C ) Samples from the cell lysates (L) and extracellular vesicles (EVs) were subjected to electrophoresis, blotted, and the membranes probed with antibodies against Hsp70, MFG-E8 and CD63. Bands were visualized by incubation with appropriate horseradish peroxidase-conjugated secondary antibodies and chemiluminescence substrate. Full blots are available in the .

Article Snippet: Cells were then incubated with a pair of primary antibodies against MFG-E8 (Santa Cruz Biotechnology; sc-217574) and P2X4 (Alomone Labs; APR-002) (dilution 1:500) for 1 h at RT, washed two times for 5 min with wash buffer A, and incubated with a mix of PLA anti-rabbit PLUS and PLA anti-mouse MINUS oligonucleotide-conjugated secondary antibodies for 1 h at 37 °C.

Techniques: Isolation, Transmission Assay, Electron Microscopy, Concentration Assay, Derivative Assay, Electrophoresis, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Extracellular Vesicles from Human Teeth Stem Cells Trigger ATP Release and Promote Migration of Human Microglia through P2X4 Receptor/MFG-E8-Dependent Mechanisms

doi: 10.3390/ijms222010970

Figure Lengend Snippet: EVs promote association between MFG-E8 and P2X4R proteins in human microglia. ( A ) Confocal images of proximity ligation assay (PLA) dots (red) in human microglial cells. The cells were treated with 1AU of EVs for 30 min, 1 h and 2 h before PLA was conducted. Nuclei, DAPI (blue). Scale bar = 100 μm. ( B ) PLA dots per cell were counted using the ImageJ program. Data shown represent results of 10 fields of view for each experimental group from three independent biological experiments. Data represents mean ± SEM, * p < 0.05; ** p < 0.01; **** p < 0.0001; n = 3; Kruskal–Wallis, Dunn‘s multiple comparisons test.

Article Snippet: Cells were then incubated with a pair of primary antibodies against MFG-E8 (Santa Cruz Biotechnology; sc-217574) and P2X4 (Alomone Labs; APR-002) (dilution 1:500) for 1 h at RT, washed two times for 5 min with wash buffer A, and incubated with a mix of PLA anti-rabbit PLUS and PLA anti-mouse MINUS oligonucleotide-conjugated secondary antibodies for 1 h at 37 °C.

Techniques: Proximity Ligation Assay

Journal: International Journal of Molecular Sciences

Article Title: Extracellular Vesicles from Human Teeth Stem Cells Trigger ATP Release and Promote Migration of Human Microglia through P2X4 Receptor/MFG-E8-Dependent Mechanisms

doi: 10.3390/ijms222010970

Figure Lengend Snippet: Co-immunoprecipitation of MFG-E8 and P2X4. Representative Western blots showing co-immunoprecipitation of MFG-E8 and P2X4R proteins in human microglial cells treated (or not) with EVs for 2 h, + indicates treatment with appropriate antibody. Full blots are available in .

Article Snippet: Cells were then incubated with a pair of primary antibodies against MFG-E8 (Santa Cruz Biotechnology; sc-217574) and P2X4 (Alomone Labs; APR-002) (dilution 1:500) for 1 h at RT, washed two times for 5 min with wash buffer A, and incubated with a mix of PLA anti-rabbit PLUS and PLA anti-mouse MINUS oligonucleotide-conjugated secondary antibodies for 1 h at 37 °C.

Techniques: Immunoprecipitation, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Extracellular Vesicles from Human Teeth Stem Cells Trigger ATP Release and Promote Migration of Human Microglia through P2X4 Receptor/MFG-E8-Dependent Mechanisms

doi: 10.3390/ijms222010970

Figure Lengend Snippet: Inhibition of MFG-E8 receptor with cilengitide suppressed EV-induced migration of microglia. For migration experiments (see above) microglial cells were pre-treated for 30 min with inhibitor MFG-E8 receptor cilengitide (10 µM), then treated with 1 AU of EVs. Data represents mean ± SD, ** p < 0.01, *** p < 0.001; n = 3; one-way ANOVA, Tukey’s multiple comparisons test.

Article Snippet: Cells were then incubated with a pair of primary antibodies against MFG-E8 (Santa Cruz Biotechnology; sc-217574) and P2X4 (Alomone Labs; APR-002) (dilution 1:500) for 1 h at RT, washed two times for 5 min with wash buffer A, and incubated with a mix of PLA anti-rabbit PLUS and PLA anti-mouse MINUS oligonucleotide-conjugated secondary antibodies for 1 h at 37 °C.

Techniques: Inhibition, Migration

Journal: International Journal of Molecular Sciences

Article Title: Extracellular Vesicles from Human Teeth Stem Cells Trigger ATP Release and Promote Migration of Human Microglia through P2X4 Receptor/MFG-E8-Dependent Mechanisms

doi: 10.3390/ijms222010970

Figure Lengend Snippet: Inhibition of MFG-E8 receptor with cilengitide suppressed EV-induced formation of lipid rafts. ( A ) Confocal images of lipid rafts (red) expressing human microglial cells. The cells were pre-treated with 10 μM cilengitide for 2 h and then treated with EVs (1 AU) of EVs for 30 min before lipid raft labeling and fixation. Lipid rafts were labeled with Vybrant ® Alexa Fluor ® 594 Lipid Raft Labeling Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. Nuclei, DAPI (blue). Scale bar = 100 μm. ( B ) The mean fluorescence intensity of lipid rafts per cell were measured with Leica Application Suite X (LAS X) software. Data shown represent the results of 15 fields of view for each experimental group from three independent biological experiments ( n = 3), plotted as the mean ± SEM. Statistical significance was analyzed by Kruskal–Wallis test using Dunn’s multiple comparison post-hoc test, *** p < 0.001; xxx p < 0.001.

Article Snippet: Cells were then incubated with a pair of primary antibodies against MFG-E8 (Santa Cruz Biotechnology; sc-217574) and P2X4 (Alomone Labs; APR-002) (dilution 1:500) for 1 h at RT, washed two times for 5 min with wash buffer A, and incubated with a mix of PLA anti-rabbit PLUS and PLA anti-mouse MINUS oligonucleotide-conjugated secondary antibodies for 1 h at 37 °C.

Techniques: Inhibition, Expressing, Labeling, Fluorescence, Software, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Extracellular Vesicles from Human Teeth Stem Cells Trigger ATP Release and Promote Migration of Human Microglia through P2X4 Receptor/MFG-E8-Dependent Mechanisms

doi: 10.3390/ijms222010970

Figure Lengend Snippet: Proposed mechanism for EV action on microglial cells. EVs carrying MFG-E8 proteins associated with phosphatidylserine exposed on the outer membrane are recognized by the αVβ3/αVβ5 integrin receptors of microglial cells and trigger lipid raft formation, interaction with P2X4 receptors, and possibly other molecules enriched in the lipid rafts such as components of the TLR4 multireceptor complex. These events lead to the upregulation of intracellular Ca 2+ , release of ATP, and increased motility of microglia.

Article Snippet: Cells were then incubated with a pair of primary antibodies against MFG-E8 (Santa Cruz Biotechnology; sc-217574) and P2X4 (Alomone Labs; APR-002) (dilution 1:500) for 1 h at RT, washed two times for 5 min with wash buffer A, and incubated with a mix of PLA anti-rabbit PLUS and PLA anti-mouse MINUS oligonucleotide-conjugated secondary antibodies for 1 h at 37 °C.

Techniques: Membrane

Journal: Molecular and Cellular Biology

Article Title: Mutual Balance of Histone Deacetylases 1 and 2 and the Acetyl Reader ATAD2 Regulates the Level of Acetylation of Histone H4 on Nascent Chromatin of Human Cells

doi: 10.1128/MCB.00421-19

Figure Lengend Snippet: Sequence analysis of individual hdac -null clones a

Article Snippet: Antibodies were as follows: mouse anti-HDAC1 (catalog no. 5356; Cell Signaling), rabbit anti-HDAC1 (catalog no. NB100-56340SS; Novus Biologicals), mouse anti-HDAC2 (catalog no. 5113; Cell Signaling), rabbit anti-HDAC2 (catalog no. 57156; Cell Signaling), anti-PCNA (catalog no. sc-56; Santa Cruz), antinucleolin (catalog no. 396400; Life Technologies), rabbit anti-ATAD2 (catalog no. 61369; Active Motif), rabbit anti-ATAD2 (catalog no. 23894-AP; Proteintech), antibiotin (mouse) (catalog no. MB-9100; Vector Laboratories), antibiotin (rabbit) (catalog no. A150-109A; Bethyl), anti-H4K12ac (catalog no. 39165; Active Motif), anti-H4K5ac (catalog no. 39699; Active Motif), anti-H3K9ac (catalog no. 9649; Cell Signaling); rabbit anti-BrdU (catalog no. GTX128091; GeneTex), rat anti-BrdU/CldU (catalog no. NB500-169; Novus Biologicals), mouse anti-BrdU/IdU (catalog no. 347580; BD Biosciences), mouse anti-RNA-DNA hybrid (clone S9.6) (catalog no. MABE1095; Millipore-Sigma), rabbit anti-RNA-DNA hybrid (clone S9.6) (catalog no. Ab01137-23.0; Absolute Antibody), rabbit anti-RNase H1 (catalog no. 15606-1-AP; Proteintech), and goat anti-V5–fluorescein isothiocyanate (FITC) (catalog no. A190-119F; Bethyl).

Techniques: Sequencing, Clone Assay

Journal: Molecular and Cellular Biology

Article Title: Mutual Balance of Histone Deacetylases 1 and 2 and the Acetyl Reader ATAD2 Regulates the Level of Acetylation of Histone H4 on Nascent Chromatin of Human Cells

doi: 10.1128/MCB.00421-19

Figure Lengend Snippet: The deletion of HDAC1 is associated with a compensatory increase in the abundance of HDAC2, and vice versa. (A) Clones of GM639 fibroblasts derived using lentiviral CRISPR-Cas9 and dgRNA vectors were seeded into a 96-well plate at the three indicated densities (from 1,000 to 4,000 cells per well) in triplicates, grown for 3 days, and subjected to a CellTiter Glo assay. Absorbance values from the assay were averaged and normalized to the value for the universal control (UC), i.e., GM639 cells with the integrated Cas9-expressing cassette and empty vector cassette for dgRNA expression (GM639-Cas9-EV). The P value shown is an example of several significant differences. (B) Representative Western blots demonstrating the absence of expression of a disrupted HDAC and a compensatory increase in the level of the remaining HDAC in the same sample. (C) HDAC2 and HDAC1 levels quantified in Western blots from multiple independent experiments were normalized to the corresponding levels in HDAC+ controls. Diamond symbols are means, and asterisks denote statistically significant differences. (D) Extracts of cells harvested in 3 independent experiments were fractionated into nucleoplasm and chromatin as described in Materials and Methods and subjected to Western blotting. Levels of HDAC1 and HDAC2 in chromatin were calculated as fractions of total levels, i.e., of the sum of the levels in chromatin and the nucleoplasm. The derived values for HDAC2 (left) and HDAC1 (right) are expressed as fold changes over values measured for HDAC+ controls. Diamond symbols are means. All P values for panels A through D were calculated by 2-sided, paired t tests. The changes observed in panel D did not reach statistical significance.

Article Snippet: Antibodies were as follows: mouse anti-HDAC1 (catalog no. 5356; Cell Signaling), rabbit anti-HDAC1 (catalog no. NB100-56340SS; Novus Biologicals), mouse anti-HDAC2 (catalog no. 5113; Cell Signaling), rabbit anti-HDAC2 (catalog no. 57156; Cell Signaling), anti-PCNA (catalog no. sc-56; Santa Cruz), antinucleolin (catalog no. 396400; Life Technologies), rabbit anti-ATAD2 (catalog no. 61369; Active Motif), rabbit anti-ATAD2 (catalog no. 23894-AP; Proteintech), antibiotin (mouse) (catalog no. MB-9100; Vector Laboratories), antibiotin (rabbit) (catalog no. A150-109A; Bethyl), anti-H4K12ac (catalog no. 39165; Active Motif), anti-H4K5ac (catalog no. 39699; Active Motif), anti-H3K9ac (catalog no. 9649; Cell Signaling); rabbit anti-BrdU (catalog no. GTX128091; GeneTex), rat anti-BrdU/CldU (catalog no. NB500-169; Novus Biologicals), mouse anti-BrdU/IdU (catalog no. 347580; BD Biosciences), mouse anti-RNA-DNA hybrid (clone S9.6) (catalog no. MABE1095; Millipore-Sigma), rabbit anti-RNA-DNA hybrid (clone S9.6) (catalog no. Ab01137-23.0; Absolute Antibody), rabbit anti-RNase H1 (catalog no. 15606-1-AP; Proteintech), and goat anti-V5–fluorescein isothiocyanate (FITC) (catalog no. A190-119F; Bethyl).

Techniques: Clone Assay, Derivative Assay, CRISPR, Glo Assay, Expressing, Plasmid Preparation, Western Blot

Journal: Molecular and Cellular Biology

Article Title: Mutual Balance of Histone Deacetylases 1 and 2 and the Acetyl Reader ATAD2 Regulates the Level of Acetylation of Histone H4 on Nascent Chromatin of Human Cells

doi: 10.1128/MCB.00421-19

Figure Lengend Snippet: A proximity ligation assay (PLA) demonstrates enrichment of the remaining HDAC on the nascent chromatin of cells with a single, hdac1- or hdac2-null mutation. (A) Diagram of the PLA approach used in the study. Cells are labeled with 10 to 20 μM EdU for 30 min and fixed. (B through M) EdU is clicked to either biotin-azide only (B through L) or a mix of biotin-azide and Alexa Fluor 488-azide at a molar ratio of 40:1 (M). (B) Representative image of HDAC2-EdU PLA foci in wild-type cells. Red fluorescence was reserved for the PLA throughout this study. (C) Numbers of HDAC2-EdU foci per nucleus were quantified in a representative experiment and plotted. Nuclei with zero foci were included in this measurement to accurately represent numbers of foci in the negative control (hdac2-36 null cells). Here and elsewhere, the UC (universal control) is GM639-Cas9-EV (see Fig. 1A legend). (D) Mean intensities of red fluorescence per nucleus were plotted separately for cells with zero or one focus (background) and in cells with two or more foci of HDAC2-EdU PLA signals. Here and elsewhere, MFI indicates the mean fluorescence intensity, and a.u. indicates arbitrary units. (E) Distributions of numbers of HDAC2-EdU PLA foci per nucleus in control and hdac1-50 cells. Only cells with two or more foci are included. (F and G) Same as panels D and E above, respectively, showing the values for HDAC2-EdU PLA signals in a different HDAC1-null clone, hdac1-47. Note that the experiments represented by panels B through E were performed with rabbit anti-HDAC2 and mouse antibiotin antibodies, and data represented in panels F and G were generated with mouse anti-HDAC2 and rabbit antibiotin antibodies. (H) Representative image of HDAC1-EdU PLA foci in the wild-type control. (I) Numbers of HDAC1-EdU foci per nucleus were quantified in a representative experiment and plotted. Nuclei with zero foci were included in this measurement to accurately represent the numbers of foci in the negative control (hdac1-50 null cells). WT1 and WT2 are GM639-Cas9 cells that have received HDAC1 or HDAC2 dgRNA-expressing vectors, respectively, but upon clonal isolation were shown to express sequence-verified wild-type HDACs. (J) Mean intensities of red fluorescence per nucleus were plotted separately for cells with zero or one focus (background) and in cells with two or more foci of HDAC1-EdU PLA signals. (K) Distributions of numbers of HDAC1-EdU PLA foci per nucleus in the controls and hdac2-21 cells. Only cells with two or more foci are included. (L) Same as for panel K, performed on a separate HDAC2-null clone, hdac2-36. (M) EdU was clicked to a mix of biotin-azide and Alexa Fluor 488-azide to distinguish EdU-positive (S-phase) (red) and EdU-negative (non-S-phase) (gray) cells. The numbers of HDAC1-EdU PLA foci were plotted separately for these two subsets of cells. Blue lines are medians. P values for mean fluorescence intensities were calculated by K-S tests, and those for numbers of foci were calculated by Wilcoxon tests. The number of cells analyzed per sample for each PLA was at least 200 and typically 400 to 500, and the HDAC1-EdU and HDAC2-EdU PLA data shown represent results from 4 independent experiments each. (N and O) Native iPOND confirms elevated recruitment of HDAC1 to nascent DNA in hdac2-null cells, as shown on a Western blot (N) and by quantitation of data from 2 independent experiments (O), where the HDAC1 level in pulldowns was first normalized to PCNA levels in pulldowns and then expressed as a fold change in hdac2-null cells over the wild-type control. Red circles are means. DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: Antibodies were as follows: mouse anti-HDAC1 (catalog no. 5356; Cell Signaling), rabbit anti-HDAC1 (catalog no. NB100-56340SS; Novus Biologicals), mouse anti-HDAC2 (catalog no. 5113; Cell Signaling), rabbit anti-HDAC2 (catalog no. 57156; Cell Signaling), anti-PCNA (catalog no. sc-56; Santa Cruz), antinucleolin (catalog no. 396400; Life Technologies), rabbit anti-ATAD2 (catalog no. 61369; Active Motif), rabbit anti-ATAD2 (catalog no. 23894-AP; Proteintech), antibiotin (mouse) (catalog no. MB-9100; Vector Laboratories), antibiotin (rabbit) (catalog no. A150-109A; Bethyl), anti-H4K12ac (catalog no. 39165; Active Motif), anti-H4K5ac (catalog no. 39699; Active Motif), anti-H3K9ac (catalog no. 9649; Cell Signaling); rabbit anti-BrdU (catalog no. GTX128091; GeneTex), rat anti-BrdU/CldU (catalog no. NB500-169; Novus Biologicals), mouse anti-BrdU/IdU (catalog no. 347580; BD Biosciences), mouse anti-RNA-DNA hybrid (clone S9.6) (catalog no. MABE1095; Millipore-Sigma), rabbit anti-RNA-DNA hybrid (clone S9.6) (catalog no. Ab01137-23.0; Absolute Antibody), rabbit anti-RNase H1 (catalog no. 15606-1-AP; Proteintech), and goat anti-V5–fluorescein isothiocyanate (FITC) (catalog no. A190-119F; Bethyl).

Techniques: Proximity Ligation Assay, Mutagenesis, Labeling, Fluorescence, Negative Control, Generated, Expressing, Isolation, Sequencing, Western Blot, Quantitation Assay

Journal: Molecular and Cellular Biology

Article Title: Mutual Balance of Histone Deacetylases 1 and 2 and the Acetyl Reader ATAD2 Regulates the Level of Acetylation of Histone H4 on Nascent Chromatin of Human Cells

doi: 10.1128/MCB.00421-19

Figure Lengend Snippet: Single HDAC deficiency changes the levels of histone acetyl PTMs in nascent chromatin. (A) Example of histone H4K12ac-EdU PLA staining in EdU-positive (S-phase) (green) and EdU-negative (non-S-phase) GM639-Cas9-EV cells. The cell-containing areas of the original image were spliced to increase the cell density for demonstration purposes. (B and C) Mean fluorescence intensities per nucleus (B) and numbers of foci per nucleus (C) of the H4K12ac-EdU PLA signal in EdU-positive and EdU-negative subsets of cells of the indicated cell lines. (D and E) Numbers of foci of H4K12ac-EdU PLA signals in EdU-positive and EdU-negative subsets of cells of additional hdac1- or hdac2-null clones. (F) Example of histone H4K5ac-EdU PLA staining in EdU-positive (S-phase) (green) and EdU-negative (non-S-phase) hdac2-36 cells. (G) Numbers of H4K5ac-EdU PLA foci in EdU-positive and EdU-negative subsets of cells of the indicated cell lines. (H) Numbers of H3K9ac-EdU PLA foci in the indicated cell lines. EdU was clicked only to biotin-azide, and only the cells showing two or more foci are included in the plot. (I) Mean fluorescence intensities of H3K9ac-EdU PLA signals per nucleus in cells with zero to one and two or more foci. In all experiments shown, cells were labeled with 10 to 20 μM EdU for 30 min and fixed. Horizontal dashes in strip chart plots are medians of distributions. P values for mean fluorescence intensities were calculated by K-S tests, and those for numbers of foci were calculated by Wilcoxon tests. ns, not significant.

Article Snippet: Antibodies were as follows: mouse anti-HDAC1 (catalog no. 5356; Cell Signaling), rabbit anti-HDAC1 (catalog no. NB100-56340SS; Novus Biologicals), mouse anti-HDAC2 (catalog no. 5113; Cell Signaling), rabbit anti-HDAC2 (catalog no. 57156; Cell Signaling), anti-PCNA (catalog no. sc-56; Santa Cruz), antinucleolin (catalog no. 396400; Life Technologies), rabbit anti-ATAD2 (catalog no. 61369; Active Motif), rabbit anti-ATAD2 (catalog no. 23894-AP; Proteintech), antibiotin (mouse) (catalog no. MB-9100; Vector Laboratories), antibiotin (rabbit) (catalog no. A150-109A; Bethyl), anti-H4K12ac (catalog no. 39165; Active Motif), anti-H4K5ac (catalog no. 39699; Active Motif), anti-H3K9ac (catalog no. 9649; Cell Signaling); rabbit anti-BrdU (catalog no. GTX128091; GeneTex), rat anti-BrdU/CldU (catalog no. NB500-169; Novus Biologicals), mouse anti-BrdU/IdU (catalog no. 347580; BD Biosciences), mouse anti-RNA-DNA hybrid (clone S9.6) (catalog no. MABE1095; Millipore-Sigma), rabbit anti-RNA-DNA hybrid (clone S9.6) (catalog no. Ab01137-23.0; Absolute Antibody), rabbit anti-RNase H1 (catalog no. 15606-1-AP; Proteintech), and goat anti-V5–fluorescein isothiocyanate (FITC) (catalog no. A190-119F; Bethyl).

Techniques: Staining, Fluorescence, Clone Assay, Labeling, Stripping Membranes

Journal: Molecular and Cellular Biology

Article Title: Mutual Balance of Histone Deacetylases 1 and 2 and the Acetyl Reader ATAD2 Regulates the Level of Acetylation of Histone H4 on Nascent Chromatin of Human Cells

doi: 10.1128/MCB.00421-19

Figure Lengend Snippet: Elevated levels of histone H4K12ac in nascent chromatin of hdac2 cells are controlled by HDAC1. (A) iPOND measurement of H4K12ac levels in nascent DNA. The indicated HDAC+ and hdac2-null clones were labeled with 10 μM EdU for 30 min and harvested for nascent DNA precipitation as described by us previously (see Materials and Methods). (B) Data from four independent iPOND experiments performed as described above for panel A with two hdac2-null clones (hdac2-21 and hdac2-36) were quantified. That is, H4K12ac abundances were first quantified by normalizing values to EdU levels in inputs, and for each experiment, the derived values were then compared between wild-type and hdac2-null cells and expressed as fold changes in an hdac2-null clone over the wild type. An open diamond is a mean value. (C) Western blot showing depletion of HDAC1 in hdac2 cells using siRNA. The percentage of remaining protein is indicated below the images. NCL, nucleolin. (D and E) Mean fluorescence intensity per nucleus of H4K12ac-EdU (D) and H4K5ac-EdU (E) PLA signals in EdU-positive and EdU-negative subsets of cells of the indicated cell lines. The graphs represent data from two independent experiments each. P values were derived by K-S tests.

Article Snippet: Antibodies were as follows: mouse anti-HDAC1 (catalog no. 5356; Cell Signaling), rabbit anti-HDAC1 (catalog no. NB100-56340SS; Novus Biologicals), mouse anti-HDAC2 (catalog no. 5113; Cell Signaling), rabbit anti-HDAC2 (catalog no. 57156; Cell Signaling), anti-PCNA (catalog no. sc-56; Santa Cruz), antinucleolin (catalog no. 396400; Life Technologies), rabbit anti-ATAD2 (catalog no. 61369; Active Motif), rabbit anti-ATAD2 (catalog no. 23894-AP; Proteintech), antibiotin (mouse) (catalog no. MB-9100; Vector Laboratories), antibiotin (rabbit) (catalog no. A150-109A; Bethyl), anti-H4K12ac (catalog no. 39165; Active Motif), anti-H4K5ac (catalog no. 39699; Active Motif), anti-H3K9ac (catalog no. 9649; Cell Signaling); rabbit anti-BrdU (catalog no. GTX128091; GeneTex), rat anti-BrdU/CldU (catalog no. NB500-169; Novus Biologicals), mouse anti-BrdU/IdU (catalog no. 347580; BD Biosciences), mouse anti-RNA-DNA hybrid (clone S9.6) (catalog no. MABE1095; Millipore-Sigma), rabbit anti-RNA-DNA hybrid (clone S9.6) (catalog no. Ab01137-23.0; Absolute Antibody), rabbit anti-RNase H1 (catalog no. 15606-1-AP; Proteintech), and goat anti-V5–fluorescein isothiocyanate (FITC) (catalog no. A190-119F; Bethyl).

Techniques: Clone Assay, Labeling, Derivative Assay, Western Blot, Fluorescence

Journal: Molecular and Cellular Biology

Article Title: Mutual Balance of Histone Deacetylases 1 and 2 and the Acetyl Reader ATAD2 Regulates the Level of Acetylation of Histone H4 on Nascent Chromatin of Human Cells

doi: 10.1128/MCB.00421-19

Figure Lengend Snippet: Ectopic expression of HDAC2 reverses elevated HDAC1 and histone acetylation in nascent chromatin. (A) Western blot showing reexpression of HDAC2 in hdac2 cells from an integrated lentiviral vector. e.v., empty vector. (B) Quantitation of immunofluorescence (IF) in situ results of staining of the indicated cells for HDAC1 and HDAC2. The data in the graph are representative of results from two independent experiments. (C and D) Scatterplots of IF in situ results similar to those in panel B showing levels of HDAC1 and HDAC2 in each nucleus of hdac2-27 cells expressing the HDAC2 transgene (C) and the wild-type control, GM639-Cas9-EV (D). (E) Mean fluorescence intensities of the HDAC1-EdU PLA signals in the indicated cell populations, subsetted by the number of PLA foci. (F) Mean fluorescence intensities of histone H4K5ac-EdU (left), H3K9ac-EdU (center), and H4K12ac-EdU (right) PLA signals in the indicated cell populations subsetted by the numbers of PLA foci. Graphs represent data from 2 independent experiments each. P values were determined by K-S tests.

Article Snippet: Antibodies were as follows: mouse anti-HDAC1 (catalog no. 5356; Cell Signaling), rabbit anti-HDAC1 (catalog no. NB100-56340SS; Novus Biologicals), mouse anti-HDAC2 (catalog no. 5113; Cell Signaling), rabbit anti-HDAC2 (catalog no. 57156; Cell Signaling), anti-PCNA (catalog no. sc-56; Santa Cruz), antinucleolin (catalog no. 396400; Life Technologies), rabbit anti-ATAD2 (catalog no. 61369; Active Motif), rabbit anti-ATAD2 (catalog no. 23894-AP; Proteintech), antibiotin (mouse) (catalog no. MB-9100; Vector Laboratories), antibiotin (rabbit) (catalog no. A150-109A; Bethyl), anti-H4K12ac (catalog no. 39165; Active Motif), anti-H4K5ac (catalog no. 39699; Active Motif), anti-H3K9ac (catalog no. 9649; Cell Signaling); rabbit anti-BrdU (catalog no. GTX128091; GeneTex), rat anti-BrdU/CldU (catalog no. NB500-169; Novus Biologicals), mouse anti-BrdU/IdU (catalog no. 347580; BD Biosciences), mouse anti-RNA-DNA hybrid (clone S9.6) (catalog no. MABE1095; Millipore-Sigma), rabbit anti-RNA-DNA hybrid (clone S9.6) (catalog no. Ab01137-23.0; Absolute Antibody), rabbit anti-RNase H1 (catalog no. 15606-1-AP; Proteintech), and goat anti-V5–fluorescein isothiocyanate (FITC) (catalog no. A190-119F; Bethyl).

Techniques: Expressing, Western Blot, Plasmid Preparation, Quantitation Assay, Immunofluorescence, In Situ, Staining, Fluorescence

Journal: Molecular and Cellular Biology

Article Title: Mutual Balance of Histone Deacetylases 1 and 2 and the Acetyl Reader ATAD2 Regulates the Level of Acetylation of Histone H4 on Nascent Chromatin of Human Cells

doi: 10.1128/MCB.00421-19

Figure Lengend Snippet: Proteomics of nascent chromatin identifies H4 acetyl PTM reader proteins. (A) Data-independent acquisition (DIA) workflow for iPOND samples. (B) Comparison of two independent label-free LC-MS/MS analyses performed on iPOND samples of HDAC+, hdac1, and hdac2 cells of the GM639 background. Shown are maximum numbers of unique peptides detected for each of the indicated proteins for all three genotypes (experiment 1, Orbitrap Fusion in DDA mode; experiment 2, Q-Exactive HF in DDA and DIA modes). (C) Workflow diagram for label-free iPOND-MS and MaxQuant data for detection of ATAD2 on EdU-labeled DNA in a biotin-specific manner (i.e., pulled down exclusively via the EdU-biotin/streptavidin bead interaction) in wild-type or hdac1- or hdac2-null GM639 cells. (D) Heat map of a cluster of proteins detected in all three genotypes in a biotin-specific manner. ATAD2 is marked by an asterisk. Red, protein is detected; green, protein is absent. Shades of red reflect ion intensities. Note that the experiment was not designed to reliably detect quantitative differences between levels of proteins present on nascent DNA. (E) Sample preparation workflow for a SILAC iPOND experiment. (F) Normalized heavy (H)/light (L) ratios for the wild type (WT) (GM639-Cas9-EV) over hdac2-21 (left) or over hdac2-36 (right) cells were rank ordered and plotted. Data points corresponding to ATAD2 are highlighted in red.

Article Snippet: Antibodies were as follows: mouse anti-HDAC1 (catalog no. 5356; Cell Signaling), rabbit anti-HDAC1 (catalog no. NB100-56340SS; Novus Biologicals), mouse anti-HDAC2 (catalog no. 5113; Cell Signaling), rabbit anti-HDAC2 (catalog no. 57156; Cell Signaling), anti-PCNA (catalog no. sc-56; Santa Cruz), antinucleolin (catalog no. 396400; Life Technologies), rabbit anti-ATAD2 (catalog no. 61369; Active Motif), rabbit anti-ATAD2 (catalog no. 23894-AP; Proteintech), antibiotin (mouse) (catalog no. MB-9100; Vector Laboratories), antibiotin (rabbit) (catalog no. A150-109A; Bethyl), anti-H4K12ac (catalog no. 39165; Active Motif), anti-H4K5ac (catalog no. 39699; Active Motif), anti-H3K9ac (catalog no. 9649; Cell Signaling); rabbit anti-BrdU (catalog no. GTX128091; GeneTex), rat anti-BrdU/CldU (catalog no. NB500-169; Novus Biologicals), mouse anti-BrdU/IdU (catalog no. 347580; BD Biosciences), mouse anti-RNA-DNA hybrid (clone S9.6) (catalog no. MABE1095; Millipore-Sigma), rabbit anti-RNA-DNA hybrid (clone S9.6) (catalog no. Ab01137-23.0; Absolute Antibody), rabbit anti-RNase H1 (catalog no. 15606-1-AP; Proteintech), and goat anti-V5–fluorescein isothiocyanate (FITC) (catalog no. A190-119F; Bethyl).

Techniques: Liquid Chromatography with Mass Spectroscopy, Labeling, Sample Prep

Journal: Molecular and Cellular Biology

Article Title: Mutual Balance of Histone Deacetylases 1 and 2 and the Acetyl Reader ATAD2 Regulates the Level of Acetylation of Histone H4 on Nascent Chromatin of Human Cells

doi: 10.1128/MCB.00421-19

Figure Lengend Snippet: The ATAD2 level in nascent chromatin is affected by HDAC status. (A) Example of an EdU-dependent ATAD2-EdU PLA in GM639-Cas9-EV cells. EdU-labeled cells were clicked to a mixture of biotin- and Alexa Fluor 488-azides. Red and green channels of the same field are shown separately. (B) Western blot demonstrating siRNA-mediated depletion of ATAD2 in hdac1-50 cells and quantitation of ATAD2-EdU PLA fluorescence in the same experiment. Cells with zero foci are included in the plot. (C and D) Numbers of ATAD2-EdU PLA foci in cells of the indicated cell lines. Only cells showing two or more foci are included. (E) Numbers of ATAD2-EdU PLA foci in EdU-negative and EdU-positive subsets of cells of the indicated cell lines. Results shown in panels C through E represent data from 5 independent experiments in total. P values were calculated by Wilcoxon tests. Black lines denote medians of distributions.

Article Snippet: Antibodies were as follows: mouse anti-HDAC1 (catalog no. 5356; Cell Signaling), rabbit anti-HDAC1 (catalog no. NB100-56340SS; Novus Biologicals), mouse anti-HDAC2 (catalog no. 5113; Cell Signaling), rabbit anti-HDAC2 (catalog no. 57156; Cell Signaling), anti-PCNA (catalog no. sc-56; Santa Cruz), antinucleolin (catalog no. 396400; Life Technologies), rabbit anti-ATAD2 (catalog no. 61369; Active Motif), rabbit anti-ATAD2 (catalog no. 23894-AP; Proteintech), antibiotin (mouse) (catalog no. MB-9100; Vector Laboratories), antibiotin (rabbit) (catalog no. A150-109A; Bethyl), anti-H4K12ac (catalog no. 39165; Active Motif), anti-H4K5ac (catalog no. 39699; Active Motif), anti-H3K9ac (catalog no. 9649; Cell Signaling); rabbit anti-BrdU (catalog no. GTX128091; GeneTex), rat anti-BrdU/CldU (catalog no. NB500-169; Novus Biologicals), mouse anti-BrdU/IdU (catalog no. 347580; BD Biosciences), mouse anti-RNA-DNA hybrid (clone S9.6) (catalog no. MABE1095; Millipore-Sigma), rabbit anti-RNA-DNA hybrid (clone S9.6) (catalog no. Ab01137-23.0; Absolute Antibody), rabbit anti-RNase H1 (catalog no. 15606-1-AP; Proteintech), and goat anti-V5–fluorescein isothiocyanate (FITC) (catalog no. A190-119F; Bethyl).

Techniques: Labeling, Western Blot, Quantitation Assay, Fluorescence

Journal: Molecular and Cellular Biology

Article Title: Mutual Balance of Histone Deacetylases 1 and 2 and the Acetyl Reader ATAD2 Regulates the Level of Acetylation of Histone H4 on Nascent Chromatin of Human Cells

doi: 10.1128/MCB.00421-19

Figure Lengend Snippet: ATAD2 depletion moderately suppresses fork progression in hdac2 cells. (A) Effects of ATAD2 depletion on percent S-phase cells in cell lines grouped by genotype. (GM639-Cas9-EV, WT1, and WT2 for HDAC+; hdac1-50 and hdac1-47 for hdac1; and hdac2-21 and hdac2-36 for hdac2). Percentages of S-phase cells were determined by calculating the fraction of EdU-positive cells after EdU pulse-labeling for 20 min followed by immunofluorescence in situ. The y axis shows differences between percent S-phase values measured for cells transfected with ATAD2 siRNA and those for cells transfected with control siRNA. Data were derived from over 10 independent experiments, each measuring at least 300 cells. The P value was determined by a Wilcoxon test. (B) Representative experiment showing profiles of EdU incorporation in cells with the indicated genotypes and siRNAs. Raw MFI values of at least 300 cells per cell line/siRNA were collected from digital images and normalized as described in Materials and Methods. The left panel includes all cells, EdU negative and EdU positive, and the right panel shows only EdU-positive cells. Vertical marks are distribution means. (C) Summary of data from multiple independent experiments similar to the one shown in panel B. For each experiment, EdU incorporation value data sets were processed as follows. The K-S test D statistic was calculated as a metric of the difference between EdU incorporation of siControl and siATAD2 cells for each genotype. The results were grouped by genotype as described above for panel A and plotted. For visualization purposes, in cases where siATAD2 cells showed reduced EdU incorporation compared to siControl, the value of the D statistic was assigned a negative sign. Black circles denote highly significant differences (P < 0.01), and white circles denote differences with P values of >0.01. Lines indicate means of the D statistic distributions. (D) Experimental design to measure fork progression rates by maRTA and data from a representative maRTA experiment. Cells were labeled sequentially with CldU and IdU for 30 min, harvested, and processed as described in Materials and Methods. Lengths of CldU and IdU segments in 100 to 300 two-segment tracks corresponding to ongoing forks were measured and plotted. P values were calculated by using K-S tests. (E) Summary of data from several independent maRTA experiments. For each experiment, differences between lengths of the 1st-label (CldU) segments in ongoing forks of siControl and siATAD2 cells were expressed as a K-S test D statistic, and the D statistic values were plotted as described above for panel C for each genotype. Black circles are the values that correspond to statistically significant differences (P < 0.01) (GM639-Cas9-EV for the WT; hdac1-50, hdac1-47, hdac1-12, and hdac1-5 for hdac1; and hdac2-21 and hdac2-36 for hdac2).

Article Snippet: Antibodies were as follows: mouse anti-HDAC1 (catalog no. 5356; Cell Signaling), rabbit anti-HDAC1 (catalog no. NB100-56340SS; Novus Biologicals), mouse anti-HDAC2 (catalog no. 5113; Cell Signaling), rabbit anti-HDAC2 (catalog no. 57156; Cell Signaling), anti-PCNA (catalog no. sc-56; Santa Cruz), antinucleolin (catalog no. 396400; Life Technologies), rabbit anti-ATAD2 (catalog no. 61369; Active Motif), rabbit anti-ATAD2 (catalog no. 23894-AP; Proteintech), antibiotin (mouse) (catalog no. MB-9100; Vector Laboratories), antibiotin (rabbit) (catalog no. A150-109A; Bethyl), anti-H4K12ac (catalog no. 39165; Active Motif), anti-H4K5ac (catalog no. 39699; Active Motif), anti-H3K9ac (catalog no. 9649; Cell Signaling); rabbit anti-BrdU (catalog no. GTX128091; GeneTex), rat anti-BrdU/CldU (catalog no. NB500-169; Novus Biologicals), mouse anti-BrdU/IdU (catalog no. 347580; BD Biosciences), mouse anti-RNA-DNA hybrid (clone S9.6) (catalog no. MABE1095; Millipore-Sigma), rabbit anti-RNA-DNA hybrid (clone S9.6) (catalog no. Ab01137-23.0; Absolute Antibody), rabbit anti-RNase H1 (catalog no. 15606-1-AP; Proteintech), and goat anti-V5–fluorescein isothiocyanate (FITC) (catalog no. A190-119F; Bethyl).

Techniques: Labeling, Immunofluorescence, In Situ, Transfection, Derivative Assay

Journal: Molecular and Cellular Biology

Article Title: Mutual Balance of Histone Deacetylases 1 and 2 and the Acetyl Reader ATAD2 Regulates the Level of Acetylation of Histone H4 on Nascent Chromatin of Human Cells

doi: 10.1128/MCB.00421-19

Figure Lengend Snippet: Working model of the relationship between HDAC1, HDAC2, ATAD2, maturation of the nascent chromatin, and DNA metabolism. The open (hyperacetylated)-to-closed (hypoacetylated) dynamic in chromatin, which takes place during replication, may affect replication itself as well as local transcription. Activities and levels of HDAC1, HDAC2, and ATAD2 may delay (e.g., when the ATAD2 level is high) or speed up (e.g., when there is no ATAD2 and no HDAC2) chromatin maturation. See Discussion for more details. RNApol, RNA polymerase.

Article Snippet: Antibodies were as follows: mouse anti-HDAC1 (catalog no. 5356; Cell Signaling), rabbit anti-HDAC1 (catalog no. NB100-56340SS; Novus Biologicals), mouse anti-HDAC2 (catalog no. 5113; Cell Signaling), rabbit anti-HDAC2 (catalog no. 57156; Cell Signaling), anti-PCNA (catalog no. sc-56; Santa Cruz), antinucleolin (catalog no. 396400; Life Technologies), rabbit anti-ATAD2 (catalog no. 61369; Active Motif), rabbit anti-ATAD2 (catalog no. 23894-AP; Proteintech), antibiotin (mouse) (catalog no. MB-9100; Vector Laboratories), antibiotin (rabbit) (catalog no. A150-109A; Bethyl), anti-H4K12ac (catalog no. 39165; Active Motif), anti-H4K5ac (catalog no. 39699; Active Motif), anti-H3K9ac (catalog no. 9649; Cell Signaling); rabbit anti-BrdU (catalog no. GTX128091; GeneTex), rat anti-BrdU/CldU (catalog no. NB500-169; Novus Biologicals), mouse anti-BrdU/IdU (catalog no. 347580; BD Biosciences), mouse anti-RNA-DNA hybrid (clone S9.6) (catalog no. MABE1095; Millipore-Sigma), rabbit anti-RNA-DNA hybrid (clone S9.6) (catalog no. Ab01137-23.0; Absolute Antibody), rabbit anti-RNase H1 (catalog no. 15606-1-AP; Proteintech), and goat anti-V5–fluorescein isothiocyanate (FITC) (catalog no. A190-119F; Bethyl).

Techniques: