Journal: Neurobiology of disease

Article Title: Ethanol exposure alters Alzheimer’s-related pathology, behavior, and metabolism in APP/PS1 mice

doi: 10.1016/j.nbd.2022.105967

Figure Lengend Snippet: Chronic moderate drinking differentially alters NMDA and GABAA receptors in the cortex and hippocampus of APP/PS1 mice. a) Ethanol treatment did not alter cortical Grin2a expression in wildtype or APP/PS1 mice. b) Ethanol-treated APP/PS1 mice had higher cortical Grin2b expression compared to EtOH-treated wildtype mice. 2-way ANOVA revealed a significant treatment × genotype interaction ( p = 0.0319). c) H 2 O-treated APP/PS1 mice showed increased cortical Gabra5 expression compared to H 2 O-exposed wildtype mice (p < 0.05). This effect was lost in EtOH-exposed APP/PS1 Gabra5 mRNA levels. 2-way ANOVA revealed a significant treatment × genotype interaction ( p = 0.0249) and a trend in genotype effects ( p = 0.0723). d) Synaptic GluN2A levels was unaltered in the hippocampus of H 2 O- or EtOH-treated wildtype or APP/PS1 mice. e) Synaptic GluN2B levels was unaltered in the hippocampus of H 2 O- or EtOH-treated wildtype or APP/PS1 mice. f) Ethanol-treated wildtype mice showed increased synaptic GABA A R α5 subunit levels compared to H 2 O-treated wildtype mice. Ethanol treatment had no effect on GABAAR α5 subunit levels in APP/PS1 mice. 2-way ANOVA revealed a significant treatment × genotype effect ( p = 0.0347) and a trend in treatment effects ( p = 0.0644). Wildtype + H2O, n = 10; APP/PS1 + H2O, n = 9; Wildtype + EtOH, n = 7; APP/PS1 + EtOH, n = 8. * p < 0.05.

Article Snippet: The following primary and secondary antibodies were used for this study: APP (including CTFβ and CTFα; Invitrogen; CT695; 1:1000), BACE1 (Cell Signaling; 5606S; 1:1000), ADAM10 (Millipore; AB19026; 1:1000), IDE (Abcam; ab232216; 1:1000), GluN2A (Cell Signaling; 4025; 1:1000), GluN2B (Cell Signaling; 4212; 1:1000), GABAAR α5 (Santa Cruz; Sc393921; 1:1000), and β-actin (Millipore; MAB1501; 1:50,000), anti-mouse (Cell Signaling; 7076S; 1:5000), anti-rabbit (Cell Signaling; 7074S; 1:5000).

Techniques: Expressing

Journal: Molecular cell

Article Title: Cohesin removal reprograms gene expression upon mitotic entry

doi: 10.1016/j.molcel.2020.01.023

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: H2A pT120 (Rabbit) , Active Motif , 39391.

Techniques: Virus, Bacteria, Recombinant, Imaging, cDNA Synthesis, Purification, Sequencing, Plasmid Preparation, Software, Staining

Journal: Nature Communications

Article Title: The chromatin remodeler RSF1 controls centromeric histone modifications to coordinate chromosome segregation

doi: 10.1038/s41467-018-06377-w

Figure Lengend Snippet: RSF1 is necessary for restricting H2A-pT120 and Sgo1 to centromeres. a HeLa cells were transfected with siRNAs, and floating mitotic cells were obtained after nocodazole treatment for 4 h and subjected to metaphase chromosome spread stained with Giemsa. Quantification of sister chromatid separation in HeLa cells after depletion of RSF1 or SNF2H. Data represent mean ± SEM; ** p < 0.01 vs. control siRNA by Student’s t -test. b HeLa cells were transfected with siRNAs, and RSF1-depleted cells were subjected to chromosome spread immunostaining. Images were obtained from representative mitotic cells: Sgo1 (green), ACA (red), and DAPI (blue). The percentages of cells exhibiting the arm or centromeric localizations of Sgo1 proteins are shown. Scale bar, 5 μm. c RSF1 knockout mitotic cells were analyzed by immunofluorescence staining: H2A-pT120 (green), ACA (red), and DAPI (blue). Percentage of cells exhibiting the arm or centromeric expression level of H2A-pT120 are shown. Scale bar, 5 μm. d Localization of Bub1 on mitotic chromosomes in RSF1 WT or knockout (KO) cells. Nocodazole-treated, mitotic RSF1 WT or knockout (KO) HeLa cells were stained with DAPI and the Bub1 antibodies. The graph represents relative intensity of Bub1 against ACA at kinetochores. At least 100 kinetochores of prometaphase cells were analyzed in three independent experiments. Scale bar, 5 μm. e Metaphase chromosome spreads were stained with anti-Bub1-pS969 and anti-ACA in RSF1 WT or KO cells. Cells were stained with DAPI and the Bub1-pS969-specific antibodies. The graph represents relative intensity of Bub1-pS969 against ACA at kinetochores. At least 100 kinetochores of prometaphase cells were analyzed in three independent experiments. Scale bar, 5 μm

Article Snippet: The antibodies were used; mouse anti-RSF1 (Upstate, 1:100, #05-727), human anti-ACA (Immunovision, Inc, 1:2,000), rabbit anti-H2A K118ac (PTM biology, 1:100, PTM-173), rabbit anti-H2A pT120 (Active motif, 1:500, #39391), mouse anti-CENP-A (abcam, 1:100, ab13939), rabbit anti-POGZ (abcam, 1:100, ab171934), rabbit anti-KAT5/Tip60 (abcam, 1:100, ab23886).

Techniques: Transfection, Staining, Control, Immunostaining, Knock-Out, Immunofluorescence, Expressing

Journal: Nature Communications

Article Title: The chromatin remodeler RSF1 controls centromeric histone modifications to coordinate chromosome segregation

doi: 10.1038/s41467-018-06377-w

Figure Lengend Snippet: Acetylation of H2A-K118 suppresses H2A-T120 phosphorylation and Sgo1 deposition. a Individual in vitro kinase assays were carried out with recombinant GST-Bub1 kinase and a positional scanning peptide library consisting of 189 biotinylated substrate peptides. b Alignment of vertebrate H2A sequence with the conserved Bub1 consensus motif. c In vitro kinase assay: recombinant GST-H2A proteins purified from bacteria were incubated with the GST-Bub1 at 30 °C for 30 min in the presence of γ 32 P-ATP. Incorporation of 32 P into H2A protein was visualized by autoradiography. Coomassie blue staining demonstrated equal protein loading. d Recombinant GST-H2A proteins were incubated with Myc-tagged Sgo1 expressing mitotic whole cell lysates for 12 h at 4 °C and subjected to immunoblotting. e RSF1 WT or KO HeLa cells were treated with paclitaxel for 16 h. Mitotic cell lysates were analyzed by immunoblotting. f Mitotic RSF1 WT or KO HeLa cell lysates were immunoprecipitated using anti-H2A-pT120 or anti-H2A-K118ac antibodies. The inputs and immunoprecipitates were analyzed by western blot using anti-CENP-A or anti-POGZ. CENP-A or POGZ proteins are specifically localized on the mitotic centromeres and chromosome arms, respectively. g Mitotic HeLa cells were stained with DAPI and H2A-K118ac (green), ACA (red), and DAPI (blue) antibodies. The percentages of cells exhibiting the chromosome arm or centromeric localizations of H2A-K118 acetylation levels are shown. Scale bar, 5 μm

Article Snippet: The antibodies were used; mouse anti-RSF1 (Upstate, 1:100, #05-727), human anti-ACA (Immunovision, Inc, 1:2,000), rabbit anti-H2A K118ac (PTM biology, 1:100, PTM-173), rabbit anti-H2A pT120 (Active motif, 1:500, #39391), mouse anti-CENP-A (abcam, 1:100, ab13939), rabbit anti-POGZ (abcam, 1:100, ab171934), rabbit anti-KAT5/Tip60 (abcam, 1:100, ab23886).

Techniques: Phospho-proteomics, In Vitro, Recombinant, Sequencing, Kinase Assay, Purification, Bacteria, Incubation, Autoradiography, Staining, Expressing, Western Blot, Immunoprecipitation

Journal: Nature Communications

Article Title: The chromatin remodeler RSF1 controls centromeric histone modifications to coordinate chromosome segregation

doi: 10.1038/s41467-018-06377-w

Figure Lengend Snippet: RSF1 recruits HDAC1 to centromeres and regulate histone H2A deacetylation. a Recombinant GST-RSF1 was incubated with Flag-HDAC1 expressing asynchronously growing cells (Asy) or mitotic cells (M) for 2 h at 4 °C. GST-RSF1 bound to Flag-HDAC1 was detected by immunoblotting. Recombinant GST-RSF1 was stained with Coomassie blue. b HeLa cells were transfected with control or RSF1 siRNA, and floating mitotic cells were obtained after nocodazole treatment for 4 h and subjected to chromosome spread immunostaining. Metaphase chromosome spreads were stained with anti-HDAC1 (green), anti-ACA (red), and DAPI (blue). The graph represents relative intensity of HDAC1 against ACA at kinetochores. Prometaphase cells were analyzed in three independent experiments. Scale bar, 5 μm. c RSF1 WT or RSF1-knockout (KO) mitotic HeLa cell lysates were blotted with the indicated antibodies. d Endogenous CENP-A (centromere protein) or POGZ (chromosome arm protein) were immunoprecipitated from nocodazole arrested RSF1 WT or RSF1 knockout HeLa cells and blotted with the indicated antibodies. e Immunoblot analysis of histone H2A acetylation levels in mitotic HeLa cells. Cells were transfected with control or HDAC1 siRNA for 48 h and floating mitotic cells were analyzed by immunoblot. f HeLa cells were transfected with control or HDAC1 siRNA, and floating mitotic cells were subjected to chromosome spread immunostaining and stained with anti-H2A-K118ac and ACA antibodies. Scale bar, 5 μm. g HeLa cells were transfected with control or HDAC1 siRNA, and floating mitotic cells were subjected to chromosome spread immunostaining and stained with anti-H2A-pT120 and ACA antibodies. Scale bar, 5 μm. h Recombinant GST-H2A proteins were incubated with control or HDAC1 siRNA transfected HeLa cells. GST-H2A proteins were incubated with recombinant GST-Bub1 in the presence of γ 32 P-ATP. Incorporation of 32 P into H2A protein was visualized by autoradiography

Article Snippet: The antibodies were used; mouse anti-RSF1 (Upstate, 1:100, #05-727), human anti-ACA (Immunovision, Inc, 1:2,000), rabbit anti-H2A K118ac (PTM biology, 1:100, PTM-173), rabbit anti-H2A pT120 (Active motif, 1:500, #39391), mouse anti-CENP-A (abcam, 1:100, ab13939), rabbit anti-POGZ (abcam, 1:100, ab171934), rabbit anti-KAT5/Tip60 (abcam, 1:100, ab23886).

Techniques: Recombinant, Incubation, Expressing, Western Blot, Staining, Transfection, Control, Immunostaining, Knock-Out, Immunoprecipitation, Autoradiography

Journal: Nature Communications

Article Title: The chromatin remodeler RSF1 controls centromeric histone modifications to coordinate chromosome segregation

doi: 10.1038/s41467-018-06377-w

Figure Lengend Snippet: The RSF1-HDAC1 interaction is required for faithful chromosome segregation. a Recombinant GST-RSF1 proteins were incubated with Flag-HDAC1 expressing mitotic lysates and subjected to immunoblotting. N1: amino acids 1–627, N2: 1–871, C2: 982–1441, PHD (plant homeodomain): 628–973. b GST-RSF1 was incubated with Flag-HDAC1 deletion mutants expressing mitotic cell lysates for 1 h at 4 °C. Precipitates were subjected to immunoblotting with anti-Flag. D1: amino acids 50–482, D2: 250–482, D3: 1–325, D4: 1-430. c The RSF1-C1 WT or 5A mutant was transfected into RSF1 KO cells, and mitotic lysates were immunoprecipitated with anti-V5 antibody, followed by immunoblotting. d , e RSF1 WT or KO HeLa cells were introduced with RSF1-C1 WT or 5A mutant and treated with nocodazole for 4 h. Floating mitotic cells were subjected to chromosome spread immunostaining with anti-HDAC1 or anti-H2A-pT120 and anti-RSF1 antibodies. The percentages of cells exhibiting the arm or centromeric localizations of HDAC1 proteins or H2A-pT120 level were plotted on a graph. f HeLa cells transfected with RSF1 WT or indicated mutants in RSF1 KO cells were subjected to metaphase chromosome spread and stained with Giemsa. Quantification of the percentage of premature sister chromatid separation in HeLa cells were shown. Scale bar, 5 μm

Article Snippet: The antibodies were used; mouse anti-RSF1 (Upstate, 1:100, #05-727), human anti-ACA (Immunovision, Inc, 1:2,000), rabbit anti-H2A K118ac (PTM biology, 1:100, PTM-173), rabbit anti-H2A pT120 (Active motif, 1:500, #39391), mouse anti-CENP-A (abcam, 1:100, ab13939), rabbit anti-POGZ (abcam, 1:100, ab171934), rabbit anti-KAT5/Tip60 (abcam, 1:100, ab23886).

Techniques: Recombinant, Incubation, Expressing, Western Blot, Mutagenesis, Transfection, Immunoprecipitation, Immunostaining, Staining

Journal: Nature Communications

Article Title: The chromatin remodeler RSF1 controls centromeric histone modifications to coordinate chromosome segregation

doi: 10.1038/s41467-018-06377-w

Figure Lengend Snippet: RSF1-deficient cells suffering from sister chromatid cohesion defects are rescued by centromere targeting HDAC1. a RSF1 KO HeLa cells were transfected with centromere targeting GFP-CENP B fused-HDAC1 WT or H141A catalytic dead mutant constructs. Floating mitotic cells were obtained after nocodazole treatment for 4 h and subjected to chromosome spread immunostaining with anti-H2A-pT120 (red) antibodies. Scale bar, 5 μm. b The cells prepared as Fig. 5a were subjected to chromosome spread immunostaining with anti-Sgo1 (red), and DAPI (blue). Scale bar, 5 μm. c Immunoblotting of chromatin fractions in RSF1 KO cells after reintroduction of CENP B fused GFP-HDAC1. d Giemsa staining of the metaphase chromosome spreads prepared after transfection of GFP-CENP B fused HDAC1 WT or H141A in RSF1 KO cells. Left panel shows quantification of the percentage of premature sister chromatid separation

Article Snippet: The antibodies were used; mouse anti-RSF1 (Upstate, 1:100, #05-727), human anti-ACA (Immunovision, Inc, 1:2,000), rabbit anti-H2A K118ac (PTM biology, 1:100, PTM-173), rabbit anti-H2A pT120 (Active motif, 1:500, #39391), mouse anti-CENP-A (abcam, 1:100, ab13939), rabbit anti-POGZ (abcam, 1:100, ab171934), rabbit anti-KAT5/Tip60 (abcam, 1:100, ab23886).

Techniques: Transfection, Mutagenesis, Construct, Immunostaining, Western Blot, Staining

Journal: Nature Communications

Article Title: The chromatin remodeler RSF1 controls centromeric histone modifications to coordinate chromosome segregation

doi: 10.1038/s41467-018-06377-w

Figure Lengend Snippet: The acetyltransferase Tip60 mediates H2A-K118 acetylation. a HeLa cells were transfected with siTip60 and arrested in mitosis and mitotic cell lysates were subjected to immunoblotting. b Lysates of HeLa cells transfected with Flag-Tip60 WT or catalytic mutant of Tip60 were blotted with indicated antibodies. c Recombinant GST-H2A protein was incubated with purified GST-Tip60 in the presence or absence of Ac-CoA and NU9056, Tip60 specific inhibitor, for in vitro acetylation assay. The acetylation levels of H2A were analyzed with an anti-H2A-K118 acetylation antibody. d Recombinant GST-H2A WT or K118 mutants were incubated with purified GST-Tip60 in the presence of Ac-CoA for in vitro acetylation assay. The acetylation levels of H2A were analyzed with an anti-H2A-K118 acetylation antibody. e Mitotic HeLa cells depleted of Tip60 were stained with anti-ACA (red), anti-H2A-K118ac (green) antibodies. The percentages of cells exhibiting the arm or centromeric level of H2A-K118ac are shown. Scale bar, 5 μm. f RSF1 WT or knockout HeLa cells were transfected with control or Tip60 siRNA and stained with anti-ACA and anti-H2A-pT120 antibodies. The percentages of cells exhibiting the arm or centromeric level of H2A-pT120 are shown. Scale bar, 5 μm

Article Snippet: The antibodies were used; mouse anti-RSF1 (Upstate, 1:100, #05-727), human anti-ACA (Immunovision, Inc, 1:2,000), rabbit anti-H2A K118ac (PTM biology, 1:100, PTM-173), rabbit anti-H2A pT120 (Active motif, 1:500, #39391), mouse anti-CENP-A (abcam, 1:100, ab13939), rabbit anti-POGZ (abcam, 1:100, ab171934), rabbit anti-KAT5/Tip60 (abcam, 1:100, ab23886).

Techniques: Transfection, Western Blot, Mutagenesis, Recombinant, Incubation, Purification, In Vitro, Acetylation Assay, Staining, Knock-Out, Control

Journal: Nature Communications

Article Title: The chromatin remodeler RSF1 controls centromeric histone modifications to coordinate chromosome segregation

doi: 10.1038/s41467-018-06377-w

Figure Lengend Snippet: A proposed model for sister chromatid cohesion by RSF1. In our model, Tip60 contributes to maintaining H2A-K118ac at chromosome arms and RSF1-bound HDAC1 at centromeres counteracts it, allowing the phosphorylation of H2A at T120 by Bub1. Green circle represents H2A-T120 and red circle is its phosphorylated form (H2A-pT120). Yellow circle represents acetylation of H2A at Lys 118 (H2A-K118ac)

Article Snippet: The antibodies were used; mouse anti-RSF1 (Upstate, 1:100, #05-727), human anti-ACA (Immunovision, Inc, 1:2,000), rabbit anti-H2A K118ac (PTM biology, 1:100, PTM-173), rabbit anti-H2A pT120 (Active motif, 1:500, #39391), mouse anti-CENP-A (abcam, 1:100, ab13939), rabbit anti-POGZ (abcam, 1:100, ab171934), rabbit anti-KAT5/Tip60 (abcam, 1:100, ab23886).

Techniques: Phospho-proteomics