Journal: Canadian Respiratory Journal

Article Title: Role of Erythromycin-Regulated Histone Deacetylase-2 in Benign Tracheal Stenosis

doi: 10.1155/2020/4213807

Figure Lengend Snippet: The expression of HDAC2 in the different groups determined by immunofluorescence. Image scale bar, 100 µ m. Red fluorescence (Cy3 staining) for the detection of the target protein HDAC2, blue fluorescence (DAPI staining) for the nucleus. Data are presented as the mean ± standard deviation in the corresponding histogram. ∗ P < 0.05 vs. the control group. Control: rabbit tracheal stenosis model without treatment; NS: rabbit tracheal stenosis model treated with penicillin; ERY: rabbit tracheal stenosis model treated with erythromycin; Budesonide: rabbit tracheal stenosis model treated with budesonide; Vorinostat: rabbit tracheal stenosis model treated with vorinostat. HDAC2, histone deacetylase-2; IF, immunofluorescence.

Article Snippet: Erythromycin enteric-coated tablets (H42021990, Yichang Humanwell Pharmaceutical Co., LTD.); Vorinostat Capsules (180509, Beijing Hengrui Kangda Medical Science and Technology Development Co., Ltd.); Budesonide (AstraZeneca 8339000); Rabbit Anti-Collagen III Polyclonal Antibody (bs-10423R, Bioss); Rabbit Anti-Collagen I Polyclonal Antibody (bs-0549R, Bioss); Rabbit Anti-HDAC2 Polyclonal Antibody (bs-1813R, Bioss); Rabbit VEGF ELISA kit (MM-021001); Rabbit TGF- β 1 ELISA kit (MM-3684001); Rabbit Polyclonal Anti-VEGF (bs-1313R, Bioss, 1/500–1/2000); Rabbit Polyclonal Anti-TGF β 1 (bs-0086R, Bioss, 1/500–1/2000); Rabbit monoclonal Anti-IL-8 (ab34100, abcam, 1/1000); Rabbit Polyclonal Anti-HDAC2 (OmnimAbs, OM105905, 1/500–1/2000); fluorescence microscope (CKX53, OLYMPUS); Microplate Reader (RT-6100, Rayto); Protein vertical electrophoresis instrument (DYY-6C, Beijing 61 instrument factory); Ultra High Sensitivity Chemiluminescence Imaging System (Chemi DocTM XRS+, Bio-Rad Shanhhai Laboratories).

Techniques: Expressing, Immunofluorescence, Fluorescence, Staining, Standard Deviation, Histone Deacetylase Assay

Journal: Genes & Development

Article Title: Histone deacetylases 1 and 2 act in concert to promote the G1-to-S progression

doi: 10.1101/gad.552310

Figure Lengend Snippet: Elevated histone acetylation in MEFs lacking HDAC1 or HDAC2. (A) Experimental strategy. Primary MEFs from HDAC1F/F mice were infected with two different retroviruses: One virus expresses Cre-ERT2 and GFP, and the second virus encodes puromycin resistance and also expresses either a small hairpin miRNA against HDAC2 or a scrambled small hairpin miRNA. Following infection, stable cell pools are obtained by puromycin selection followed by FACS sorting of GFP-positive cells. Addition of tamoxifen allows us to generate the four different genotypes (wild-type [WT], HDAC1 KO, HDAC2 Kd, and HDAC1/2 KO/Kd) from the same starting cells, allowing a direct comparison. (B) Protein extracts were prepared from cells of the four different genotypes and probed with antibodies against HDAC1, HDAC2, and actin, as indicated. Western blot analysis was done using the Odyssey Infrared Imaging System. The quantification of band intensities is indicated below using arbitrary units. (Lane 1) The amount of HDAC1 or HDAC2 in the initial cells was set to 100%. (C) Loss of HDAC1 and/or HDAC2 leads to selective increase in histone acetylation. Nuclear extracts from the indicated MEF cells were probed with antibodies recognizing H3 or H4, or the acetylated forms of H3, H4, H3K14, H4K5, H4K8, H4K12, or H4K16. The signal for H3 and H4 is presented, demonstrating equal protein loading. Western blot analysis was done using the Odyssey Infrared Imaging System. The quantification of band intensities is indicated below using arbitrary units. (Lane 1) The amount in the wild-type cells was set to 100, and all values were corrected for the H3 or H4 signal. A representative experiment is presented.

Article Snippet: The antibodies used in this study were the following: HDAC1 and HDAC2 (gift from Christian Seiser, Vienna Biocenter), actin (Ab-5; Neo Markers), cyclin D2 (MBL International Corporation), cyclin B1 (MBL International Corporation), p15 (Cell Signaling), p16 (M-156, Santa Cruz Biotechnologies), p21 (F-5, Santa Cruz Biotechnologies), p27 (F-8, Santa Cruz Biotechnologies), p57 (p2735, Sigma), Co-REST (07-455, Millipore), Sin3a (06-913, Millipore), acetyl H4 (06-866, Millipore), acetyl H3 (06-599, Millipore), acetyl H3K14 (07-353, Millipore), acetyl H4K5 (07-327, Millipore), acetyl H4K8 (07-328, Millipore), and acetyl H4K12 (06-761, Millipore).

Techniques: Infection, Stable Transfection, Selection, Western Blot, Imaging

Journal: Genes & Development

Article Title: Histone deacetylases 1 and 2 act in concert to promote the G1-to-S progression

doi: 10.1101/gad.552310

Figure Lengend Snippet: Elimination of HDAC1 and HDAC2 leads to impaired cell proliferation and induction of apoptosis. (A) Growth curve. MEFs of the different genotypes were passaged every 3 or 4 d, the total number of cells was counted, and cells were reseeded at 5 × 105 cells per 100-mm dish. Tamoxifen was added at day 4. Each growth curve is the average (±SD) of three independent MEF cells generated from different embryos. (Solid black line) Wild-type cells; (black dashed line) HDAC1 KO cells; (solid gray line) HDAC2 Kd cells; (gray dashed line) HDAC1/2 KO/Kd cells. (B) Analysis of apoptosis by annexin V staining. HDAC1F/F MEF cells expressing Cre-ERT2 and a small hairpin miRNA against HDAC2 were followed over time after addition of tamoxifen. After fixation and staining with an annexin V antibody, the cells were analyzed by flow cytometry. The bar graph represents the average of three independent experiments (±SD). (C) Gene expression profiles. Supervised hierarchical clustering was performed using comparative expression values of the gene lists indicated above the dendrogram (for gene lists, see Supplemental Table 1). In all cases, the expression value for the wild-type cells was used to normalize expression levels. (D) Gene Ontology term analysis of the main gene expression changes observed in the different MEF lines. The networks and molecular functions scoring the eight highest P-value in HDAC1/2 KO/Kd cells are listed together with the P-value in each single-mutant cell.

Article Snippet: The antibodies used in this study were the following: HDAC1 and HDAC2 (gift from Christian Seiser, Vienna Biocenter), actin (Ab-5; Neo Markers), cyclin D2 (MBL International Corporation), cyclin B1 (MBL International Corporation), p15 (Cell Signaling), p16 (M-156, Santa Cruz Biotechnologies), p21 (F-5, Santa Cruz Biotechnologies), p27 (F-8, Santa Cruz Biotechnologies), p57 (p2735, Sigma), Co-REST (07-455, Millipore), Sin3a (06-913, Millipore), acetyl H4 (06-866, Millipore), acetyl H3 (06-599, Millipore), acetyl H3K14 (07-353, Millipore), acetyl H4K5 (07-327, Millipore), acetyl H4K8 (07-328, Millipore), and acetyl H4K12 (06-761, Millipore).

Techniques: Generated, Staining, Expressing, Flow Cytometry, Mutagenesis

Journal: Genes & Development

Article Title: Histone deacetylases 1 and 2 act in concert to promote the G1-to-S progression

doi: 10.1101/gad.552310

Figure Lengend Snippet: Loss of HDAC1 and HDAC2 leads to a cell cycle block in G1 accompanied by increased levels of CDKis p21WAF1/CIP1 and p57Kip2. (A) Cell cycle analysis by flow cytometry. Following a 30-min pulse of BrdU, cells were collected and ethanol-fixed. After staining with an antibody against BrdU and 7-AAD, cells were analyzed by FACS. Representative dot plots are shown for each genotype, and the percentage of cells in the different cell cycle phases are indicated to the right of each panel. (B) Protein extracts were prepared from wild-type, HDAC1 KO, HDAC2 Kd, and HDAC1/2 KO/Kd cells and probed with antibodies against cyclin D2, cyclin B1, and actin, as indicated. (C) Deregulated expression of CDKis in the absence of HDAC1 and HDAC2. Protein extracts were prepared from wild-type, HDAC1 KO, HDAC2 Kd, and HDAC1/2 KO cells and probed with antibodies against p15 (cdkn2b), p16 (cdkn2a), p21 (cdkn1a), p27 (cdkn1b), and p57 (cdkn1c), as indicated. (D) p21 protein expression increases proportionally to the down-regulation of HDAC1. Deletion of HDAC1 was induced in HDAC2 Kd cells by addition of tamoxifen, and the level of HDAC1, HDAC2, and p21 proteins was followed by Western blot analysis at different time points, as indicated. Band intensities were quantified using the Odyssey Infrared Imaging System and were plotted using arbitrary units.

Article Snippet: The antibodies used in this study were the following: HDAC1 and HDAC2 (gift from Christian Seiser, Vienna Biocenter), actin (Ab-5; Neo Markers), cyclin D2 (MBL International Corporation), cyclin B1 (MBL International Corporation), p15 (Cell Signaling), p16 (M-156, Santa Cruz Biotechnologies), p21 (F-5, Santa Cruz Biotechnologies), p27 (F-8, Santa Cruz Biotechnologies), p57 (p2735, Sigma), Co-REST (07-455, Millipore), Sin3a (06-913, Millipore), acetyl H4 (06-866, Millipore), acetyl H3 (06-599, Millipore), acetyl H3K14 (07-353, Millipore), acetyl H4K5 (07-327, Millipore), acetyl H4K8 (07-328, Millipore), and acetyl H4K12 (06-761, Millipore).

Techniques: Blocking Assay, Cell Cycle Assay, Flow Cytometry, Staining, Expressing, Western Blot, Imaging

Journal: Genes & Development

Article Title: Histone deacetylases 1 and 2 act in concert to promote the G1-to-S progression

doi: 10.1101/gad.552310

Figure Lengend Snippet: HDAC1 and HDAC2 are bound to the p21 and p57 promoters and regulate their expression. (A) Analysis of HDAC binding to the p21 and p57 promoters by ChIP assays. ChIP was performed for either HDAC1 or HDAC2 in wild-type, HDAC1 KO, and HDAC2 Kd cells, as indicated. Enrichment was assessed by qPCR for the p21 and p57 promoters normalized to the amplification obtained with the actin promoter. Results presented are an average of three independent experiments (±SD), with each PCR amplification performed in triplicate. (B) Deregulated expression of the p21 and p57 genes in the absence of HDAC1 and HDAC2. RNA was extracted from the different cells as indicated, and was used for qRT–PCR assays. The expression level of p21 or p57 in wild-type cells was set to 100%, after normalization by measuring the expression of Hprt1. The average (±SD) from two independent experiments is presented.

Article Snippet: The antibodies used in this study were the following: HDAC1 and HDAC2 (gift from Christian Seiser, Vienna Biocenter), actin (Ab-5; Neo Markers), cyclin D2 (MBL International Corporation), cyclin B1 (MBL International Corporation), p15 (Cell Signaling), p16 (M-156, Santa Cruz Biotechnologies), p21 (F-5, Santa Cruz Biotechnologies), p27 (F-8, Santa Cruz Biotechnologies), p57 (p2735, Sigma), Co-REST (07-455, Millipore), Sin3a (06-913, Millipore), acetyl H4 (06-866, Millipore), acetyl H3 (06-599, Millipore), acetyl H3K14 (07-353, Millipore), acetyl H4K5 (07-327, Millipore), acetyl H4K8 (07-328, Millipore), and acetyl H4K12 (06-761, Millipore).

Techniques: Expressing, Binding Assay, Amplification, Quantitative RT-PCR

Journal: Genes & Development

Article Title: Histone deacetylases 1 and 2 act in concert to promote the G1-to-S progression

doi: 10.1101/gad.552310

Figure Lengend Snippet: In vivo early B-cell development requires HDAC1 or HDAC2. (A) Scheme of early B-cell differentiation, depicting the different developmental stages and the expression of markers used to discriminate them: Marker expression is indicated by (+), lack of expression is indicated by (−). Curved arrows indicate relative cellular proliferation activities: very high in large pre-BII, intermediate in pro-BI and pre-BI, and low or absent in small pre-BII and immature and mature B cells. The expression of immunoglobulin (IgM) at the membrane is also depicted. At the top of the scheme, the pattern of cre expression in mb1-cre and CD23-cre mice is presented. (B) Western blot analysis of HDAC1 or HDAC2 in B cells of HDAC1F/F or HDAC2F/F + mb1-cre mice. Protein extracts from splenic mature B cells were probed with antibodies against HDAC1, HDAC2, or actin, as indicated. (C) Flow cytometric analysis of single-KO (HDAC1F/F + mb1-cre or HDAC2F/F + mb1-cre) or double-KO (HDAC1/2F/F + mb1-cre) mice. Single-cell suspensions from spleen or bone marrow were stained with the indicated antibody combinations and analyzed by FACS. Individual dot plots are shown that are representative of more than three independent experiments. Numbers in plots represent the percentage of cells in the respective quadrant. A simplified scheme of the B-cell developmental progression based on the B220 and IgM markers (X-axis/Y-axis) is shown on the right. (D) Total cell numbers present in different B-cell populations in the bone marrow. The absolute numbers were determined based on FACS analysis and cell counting. The histograms represent the mean ± SE based on the analysis of at least four mice per genotype.

Article Snippet: The antibodies used in this study were the following: HDAC1 and HDAC2 (gift from Christian Seiser, Vienna Biocenter), actin (Ab-5; Neo Markers), cyclin D2 (MBL International Corporation), cyclin B1 (MBL International Corporation), p15 (Cell Signaling), p16 (M-156, Santa Cruz Biotechnologies), p21 (F-5, Santa Cruz Biotechnologies), p27 (F-8, Santa Cruz Biotechnologies), p57 (p2735, Sigma), Co-REST (07-455, Millipore), Sin3a (06-913, Millipore), acetyl H4 (06-866, Millipore), acetyl H3 (06-599, Millipore), acetyl H3K14 (07-353, Millipore), acetyl H4K5 (07-327, Millipore), acetyl H4K8 (07-328, Millipore), and acetyl H4K12 (06-761, Millipore).

Techniques: In Vivo, Cell Differentiation, Expressing, Marker, Western Blot, Staining, Cell Counting

Journal: Genes & Development

Article Title: Histone deacetylases 1 and 2 act in concert to promote the G1-to-S progression

doi: 10.1101/gad.552310

Figure Lengend Snippet: Ablation of HDAC1 and HDAC2 in vivo induces a severe cell cycle block in G1 and apoptosis. (A) In vivo BrdU incorporation assay. Large pre-BII cells from HDAC1/2F/F + mb1-cre or control (HDAC1/2F/F) mice were sorted by FACS as indicated in the Materials and Methods. After fixation, permeabilization, and DNA digestion, the DNA was stained with an anti-BrdU antibody together with 7-AAD, and analysis was done by flow cytometry. Numbers in plots indicate the percentage of cells in each cell cycle phase. On the right, a schematic is presented in which the different cell populations are labeled. (B) Analysis of apoptosis by annexin V staining. Cells were stained for cell surface markers and annexin V, and were analyzed by flow cytometry. Data are representative of more than three mice of each genotype.

Article Snippet: The antibodies used in this study were the following: HDAC1 and HDAC2 (gift from Christian Seiser, Vienna Biocenter), actin (Ab-5; Neo Markers), cyclin D2 (MBL International Corporation), cyclin B1 (MBL International Corporation), p15 (Cell Signaling), p16 (M-156, Santa Cruz Biotechnologies), p21 (F-5, Santa Cruz Biotechnologies), p27 (F-8, Santa Cruz Biotechnologies), p57 (p2735, Sigma), Co-REST (07-455, Millipore), Sin3a (06-913, Millipore), acetyl H4 (06-866, Millipore), acetyl H3 (06-599, Millipore), acetyl H3K14 (07-353, Millipore), acetyl H4K5 (07-327, Millipore), acetyl H4K8 (07-328, Millipore), and acetyl H4K12 (06-761, Millipore).

Techniques: In Vivo, Blocking Assay, BrdU Incorporation Assay, Staining, Flow Cytometry, Labeling

Journal: Genes & Development

Article Title: Histone deacetylases 1 and 2 act in concert to promote the G1-to-S progression

doi: 10.1101/gad.552310

Figure Lengend Snippet: HDAC1 and HDAC2 are dispensable in mature resting B cells, but are required for their proliferation. (A) Flow cytometric analysis of double-KO (HDAC1/2F/F + CD23-cre) and control (HDAC1/2F/F) mice. Single-cell suspensions from spleen were stained with the indicated antibody combination and analyzed by FACS. The dot plots presented are representative of an analysis done with four mice of each genotype. Numbers in plots represent the percentage of cells in the respective quadrant. (B) Mature B cells lacking HDAC1 and HDAC2 fail to proliferate in vitro. Splenic B cells were isolated from double-KO or control mice, stained with CFSE, and induced to proliferate in vitro by addition of LPS + IL-4. The distribution of CFSE-positive cells was analyzed by FACS after 72 h of stimulation. (C) Apoptosis induction in stimulated cells. Cells were stained with CFSE, stimulated for 72 h as above, and analyzed by annexin V staining.

Article Snippet: The antibodies used in this study were the following: HDAC1 and HDAC2 (gift from Christian Seiser, Vienna Biocenter), actin (Ab-5; Neo Markers), cyclin D2 (MBL International Corporation), cyclin B1 (MBL International Corporation), p15 (Cell Signaling), p16 (M-156, Santa Cruz Biotechnologies), p21 (F-5, Santa Cruz Biotechnologies), p27 (F-8, Santa Cruz Biotechnologies), p57 (p2735, Sigma), Co-REST (07-455, Millipore), Sin3a (06-913, Millipore), acetyl H4 (06-866, Millipore), acetyl H3 (06-599, Millipore), acetyl H3K14 (07-353, Millipore), acetyl H4K5 (07-327, Millipore), acetyl H4K8 (07-328, Millipore), and acetyl H4K12 (06-761, Millipore).

Techniques: Staining, In Vitro, Isolation