Journal: Journal of Neuroscience

Article Title: Regulation of p27Kip1 by Sox2 Maintains Quiescence of Inner Pillar Cells in the Murine Auditory Sensory Epithelium

doi: 10.1523/jneurosci.0686-12.2012

Figure Lengend Snippet: Figure 1. Deletion of Sox2 leads to proliferation of neonatal IPCs. A–F, Triple staining of myosin VI, Sox2, and EdU in Fgfr3iCreER; Sox2/ control (A–C) and Fgfr3iCreER; Sox2loxP/loxP experimental (D–F) samples given tamoxifen at P0 and P1 at theHClayer(A,D)andSClayer(B,E).CandFareartificialcross-sectionimagesintheYZplane.ArrowsinEandFpointtothesame EdU/Sox2-negative IPC. Dashed lines in C and F represent the basilar membrane. G–H, Control (G) and experimental (H–H) sampleswerestainedwithmyosinVI,EdU,andBrdU.GandHareimagestakenattheHClayer,andHattheSClayer.ArrowinH showsanEdU/BrdUIPC.I,QuantificationofEdUIPCsintheentirecochleaofexperimentalsamplesgivenTMXatP0andP1, EdU at P2, and analyzed 6 h later or at P4.**p 0.01 (n 3). Scale bars, 20 m.

Article Snippet: Next DNA/protein complexes were immunoprecipitated using 2 l of ChIP-formulated Sox2 antibody (Cell Signaling Technology, 5024).

Techniques: Staining, Control, Membrane

Journal: Journal of Neuroscience

Article Title: Regulation of p27Kip1 by Sox2 Maintains Quiescence of Inner Pillar Cells in the Murine Auditory Sensory Epithelium

doi: 10.1523/jneurosci.0686-12.2012

Figure Lengend Snippet: Figure 2. Deletion of Sox2 results in loss of p27Kip1 expression in neonatal IPCs without cell fate change. A, Quantification of Sox2- negativeandp27Kip1-negativeIPCsatP2intheentirecochleaofFgfr3iCreER;Sox2loxP/loxPexperimentalmiceinjectedwithtamoxifenatP0 andP1.B–B,TriplestainingofSox2,p27Kip1,andEdUincochlearsamplesfromexperimentalmice.ArrowspointtothesameEdU/ Sox2-negative/p27Kip1-negativeIPC.ArrowheadspointtothesameSox2-negativeIPCthatmaintainedfaintexpressionofp27Kip1 and was EdU-negative, which is also visualized at a higher magnification (inset in B). C–D, Double labeling of Sox2 and Prox1 in control Fgfr3iCreER;Sox2/(C–C)andexperimentalFgfr3iCreER;Sox2loxP/loxP(D–D)samples.ImageswerevisualizedatconfocalYZplane. MoreIPCswerepresentinexperimental(dashedcircleinD)thanincontrolgroups.Scalebars,20m.

Article Snippet: Next DNA/protein complexes were immunoprecipitated using 2 l of ChIP-formulated Sox2 antibody (Cell Signaling Technology, 5024).

Techniques: Expressing, Labeling, Control

Journal: Journal of Neuroscience

Article Title: Regulation of p27Kip1 by Sox2 Maintains Quiescence of Inner Pillar Cells in the Murine Auditory Sensory Epithelium

doi: 10.1523/jneurosci.0686-12.2012

Figure Lengend Snippet: Figure 3. Sox2 ablation causes a loss of p27Kip1 expression and S phase reentry of juvenile IPCs. A–B, Fgfr3iCreER; Sox2/

Article Snippet: Next DNA/protein complexes were immunoprecipitated using 2 l of ChIP-formulated Sox2 antibody (Cell Signaling Technology, 5024).

Techniques: Expressing

Journal: Journal of Neuroscience

Article Title: Regulation of p27Kip1 by Sox2 Maintains Quiescence of Inner Pillar Cells in the Murine Auditory Sensory Epithelium

doi: 10.1523/jneurosci.0686-12.2012

Figure Lengend Snippet: Figure4. Sox2-negativeIPCsinjuvenilemicecannotcompletetheentirecellcycleandmaintainedaSCfate.A–B ,TriplestainingofSox2,pH3andEdUinsamplesfromFgfr3iCreER;Sox2loxP/loxP

Article Snippet: Next DNA/protein complexes were immunoprecipitated using 2 l of ChIP-formulated Sox2 antibody (Cell Signaling Technology, 5024).

Techniques:

Journal: Journal of Neuroscience

Article Title: Regulation of p27Kip1 by Sox2 Maintains Quiescence of Inner Pillar Cells in the Murine Auditory Sensory Epithelium

doi: 10.1523/jneurosci.0686-12.2012

Figure Lengend Snippet: Figure7. Proliferatingp27 Kip1-nullPCsmaintainProx1andSox2expression.A–A,Double labeling of Sox2 and p27 Kip1 at P2 in Prox1CreER/; p27loxP/loxP (experimental) samples given tamoxifenatP0andP1.ArrowspointtothesameSox2/p27 Kip1-negativeIPC.B–B,Cross- section staining of EdU and Prox1 at P2. Arrows point to the same Prox1/ EdU PC. Similar results were observed at P4. Scale bars, 20 m.

Article Snippet: Next DNA/protein complexes were immunoprecipitated using 2 l of ChIP-formulated Sox2 antibody (Cell Signaling Technology, 5024).

Techniques: Labeling, Staining

Journal: Journal of Neuroscience

Article Title: Regulation of p27Kip1 by Sox2 Maintains Quiescence of Inner Pillar Cells in the Murine Auditory Sensory Epithelium

doi: 10.1523/jneurosci.0686-12.2012

Figure Lengend Snippet: Figure 8. Long-term effects caused by proliferation of neonatal IPCs. A–B, Whole-mount image of calbindin HCs in Fgfr3iCreER; Sox2/ control (A, A) and Fgfr3iCreER; Sox2loxP/loxP experimental (B, B) groups. Arrow in B indicates 3 missed IHCs. C, Projection image of the rectangular area in B showing loss of OHCs. D, D, Image of TUNEL and calbindin labeling in experimental mice at P16. Arrows indicate dying cells. Scale bars: B, 200 m; C–D, 20 m.

Article Snippet: Next DNA/protein complexes were immunoprecipitated using 2 l of ChIP-formulated Sox2 antibody (Cell Signaling Technology, 5024).

Techniques: Control, TUNEL Assay, Labeling

Journal: Journal of Neuroscience

Article Title: Regulation of p27Kip1 by Sox2 Maintains Quiescence of Inner Pillar Cells in the Murine Auditory Sensory Epithelium

doi: 10.1523/jneurosci.0686-12.2012

Figure Lengend Snippet: Figure9. Sox2regulatesp27 Kip1invitro.A,Schematicoftheluciferaseconstructusedinthereporterassay.Theblueregionis a 3.8 kb putative p27kip1 promoter fragment. B–D, The effect of Sox2 overexpression on p27Kip1 transcriptional activity was measured in MEF (B), HeLa (C), and HEK cells (D). All values were normalized to the negative control empty vector (EV), then compared with the positive control E2F1, or Sox2 overexpression. Minimal luciferase activity was detected when a promoter-less luciferase vector was used (empty-luc), with no increases occurring in the presence of Sox2 or E2F1. E, Schematic of the p27- luciferase plasmid and amplicon location. F, qPCR data from ChIP experiments performed in MEF cells transfected with p27- luciferaseplasmidandSox2.Asignificantenrichmentofamplicon7(1400bpupstreamofLuciferaseORF)wasobservedwhen the Sox2 antibody was used for ChIP. *p 0.05.

Article Snippet: Next DNA/protein complexes were immunoprecipitated using 2 l of ChIP-formulated Sox2 antibody (Cell Signaling Technology, 5024).

Techniques: Over Expression, Activity Assay, Negative Control, Plasmid Preparation, Positive Control, Luciferase, Amplification, Transfection

Journal: Frontiers in Immunology

Article Title: Protective Immunity Induced by Incorporating Multiple Antigenic Proteins of Toxoplasma gondii Into Influenza Virus-Like Particles

doi: 10.3389/fimmu.2018.03073

Figure Lengend Snippet: Diagrams for multi-antigen VLPs (TG146 VLPs, A ) and combination VLPs (TG1/TG4/TG6 VLPs, B ). Multi-antigen VLPs is a vaccine formulation in which VLPs are generated by simultaneously infecting Sf9 cells with three baculoviruses expressing TG1, TG4, and TG6 proteins together with influenza M1. Combination VLPs is a vaccine formulation in which the exact same amounts of VLPs from TG1 VLPs, TG4 VLPs, and TG6 VLPs are combined. Western blot analyses for IMC (TG1), ROP18 (TG4), and MIC8 (TG6) from T. gondii and influenza M1 were performed to determine incorporation into VLPs (C,D) . Multi-antigen VLPs (C) and combination VLPs (D) were loaded for SDS-PAGE. Polyclonal mouse anti- T. gondii antibody and anti-M1 monoclonal antibody were used to probe T. gondii IMC (TG1), ROP18 (TG4), MIC8 (TG6) protein, and influenza M1 protein. VLPs protein concentrations for Lane 1: 27 μg, lane 2: 9 μg and, lane 3: 3 μg were used.

Article Snippet: Monoclonal mouse anti-M1 antibody was purchased from Abcam (Cambridge, UK).

Techniques: Formulation, Generated, Expressing, Western Blot, SDS Page

Journal: Cell reports

Article Title: p63 and SOX2 Dictate Glucose Reliance and Metabolic Vulnerabilities in Squamous Cell Carcinomas

doi: 10.1016/j.celrep.2019.07.027

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit monoclonal anti-Sox2 ChIP Formulated , Cell Signaling Technology , Cat# 5024; Clone D6D9; RRID: AB_1904142.

Techniques: Diagnostic Assay, Virus, Plasmid Preparation, Recombinant, Membrane, Luciferase, Enzyme-linked Immunosorbent Assay, Expressing, Cloning, shRNA, Sequencing, Software, Real-time Polymerase Chain Reaction, Microscopy, Imaging

Journal: Science Advances

Article Title: ATP-binding cassette protein ABCF1 couples transcription and genome surveillance in embryonic stem cells through low-complexity domain

doi: 10.1126/sciadv.abk2775

Figure Lengend Snippet: ( A ) Chromatography scheme for purification of SCC-B from NT2 nuclear extracts (NT2 NE). NT2 NE is first subjected to ammonium sulfate precipitation (55% saturation) followed by a series of chromatographic columns including nickel affinity agarose (Ni-NTA), cation exchangers phosphocellulose (P11), heparin (Poros-HE), Mono S, anion exchanger Poros-HQ, hydroxyapatite (HAP), and gel filtration medium Superose 6. ( B ) Input fraction containing SCC-B activity from the Poros-HE step (IN), flow-through (FT), and various salt-eluted Mono S fractions was assayed for their ability to stimulate OCT4/SOX2-dependent transcription from the human NANOG promoter template engineered with four extra copies of the oct-sox composite binding element (bottom). All reactions contain purified general transcription factors (GTFs), Pol II, OCT4, SOX2, and recombinant XPC and DKC1 complexes. Transcribed RNA products are subjected to primer extension and visualized by autoradiography. ( C ) Mono S fractions assayed in in vitro transcription are separated on a polyacrylamide gel and stained with silver. Filled arrowhead indicates the ~110-kDa polypeptide that comigrates with SCC-B transcriptional activity. ( D ) Coomassie staining of Mono S fraction 14 demonstrates purification to homogeneity. ( E ) ABCF1 is enriched in human ES cells. Down-regulation of ABCF1 in human ES cell line H9 upon exit from pluripotency. ( F ) ABCF1 is enriched in mouse ES cells. mRNA and protein levels of ABCF1 in pluripotent D3 mouse ES cells carrying V5 epitope–tagged Abcf1 alleles (V5-ABCF1 knock-in) are compared to their differentiated counterparts.

Article Snippet: Polyclonal antibodies against ABCF1 (13950-1-AP) were purchased from ProteinTech; XPC (A301-122A) and mouse Nanog (A300-397A) from Bethyl Laboratories; DKC1 (H-300), OCT4 (N-19), and Pol II (N-20) from Santa Cruz Biotechnology; SOX2 (AB5603) from EMD Millipore; V5 ChIP grade (ab15828) from Abcam; and histone H3 (ChIP formulated, no. 2650) from Cell Signaling Technology (CST).

Techniques: Chromatography, Purification, Filtration, Activity Assay, Binding Assay, Recombinant, Autoradiography, In Vitro, Staining, Knock-In

Journal: Science Advances

Article Title: ATP-binding cassette protein ABCF1 couples transcription and genome surveillance in embryonic stem cells through low-complexity domain

doi: 10.1126/sciadv.abk2775

Figure Lengend Snippet: ( A ) shRNA-mediated knockdown of ABCF1 in mouse ES cells. Asterisk denotes a nonspecific band. ( B ) Depletion of ABCF1 in mouse ES cells leads to colony collapse with flattened cell morphology and reduced alkaline phosphatase (AP) staining, indicating spontaneous differentiation. ( C ) Loss of ABCF1 in mouse ES cells compromises pluripotency gene expression. Quantification of mRNA levels of pluripotency genes is analyzed by qPCR and normalized to Actb . ( D ) Depletion of ABCF1 induces expression of genes associated with the three embryonic germ layers and the trophectoderm, analyzed by qPCR as in (C). ( E ) Depletion of ABCF1 blocks somatic cell reprogramming. AP + colonies are counted after 14 days (11 days with doxycycline followed by 3 days of dox withdrawal) post-induction (dpi). ( F ) Single-cell suspensions of 14 dpi reprogrammed CF-1 MEFs as described in (E) are stained with anti-mouse SSEA-1 and analyzed by flow cytometry. ( G ) Micrococcal nuclease (MNase) ChIP analysis of ABCF1 occupancy on control and enhancer regions of Oct4 , Sox2 , and Nanog gene loci in V5-ABCF1 knock-in D3 mouse ES cells. Representative data showing the enrichment of V5-ABCF1 (gray bars) compared to control IgGs (white bars) are analyzed by qPCR and expressed as percentage of input chromatin. Schematic diagrams of OCT4/SOX2 binding sites of each gene and the relative positions of the amplicons are shown at the bottom. ( H ) ABCF1 is not recruited to the promoter of housekeeping gene Actb .

Article Snippet: Polyclonal antibodies against ABCF1 (13950-1-AP) were purchased from ProteinTech; XPC (A301-122A) and mouse Nanog (A300-397A) from Bethyl Laboratories; DKC1 (H-300), OCT4 (N-19), and Pol II (N-20) from Santa Cruz Biotechnology; SOX2 (AB5603) from EMD Millipore; V5 ChIP grade (ab15828) from Abcam; and histone H3 (ChIP formulated, no. 2650) from Cell Signaling Technology (CST).

Techniques: shRNA, Staining, Expressing, Flow Cytometry, Knock-In, Binding Assay

Journal: Science Advances

Article Title: ATP-binding cassette protein ABCF1 couples transcription and genome surveillance in embryonic stem cells through low-complexity domain

doi: 10.1126/sciadv.abk2775

Figure Lengend Snippet: ( A ) GST fusion proteins containing the LCD of human ABCF1 (1–302), the N-terminal domain of yeast GCN20 (1–197), and the transactivation domain of human transcription factor SREBP1a (1–50) are incubated with buffer only (−) or NT2 NEs (+). Input (IN) indicated. ( B ) Whole-cell extracts (WCEs) from 293T cells cotransfected with plasmid expressing V5-tagged ABCF1 together with either empty plasmid (−) or plasmids expressing FLAG-tagged SOX2 (S) or OCT4 (O) are immunoprecipitated with anti-FLAG antibody. ( C ) Input V5-ABCF1 KI mouse ES cell WCEs (IN) and IPs by IgG and anti-SOX2 antibodies are analyzed by Western blotting. ( D ) HA IPs from 293T cells overexpressing HA-tagged SOX2 (SOX2-HA) with V5-tagged FL or LCD-truncated human ABCF1 (NBDs). ( E ) ABCF1 knockdown rescue assay. mRNAs from control mouse ES cells (shNT) overexpressing RFP, and ABCF1 knockdown ES cells (sh1) overexpressing RFP, V5-tagged FL, LCD-truncated human ABCF1 (NBDs), or mouse NANOG are analyzed for Nanog , Fgf4 , and Klf4 mRNA levels by qPCR. ( F ) Colony formation assays of cells described in (E). Differentiation status is evaluated on the basis of AP staining intensity and colony morphology. Representative images of AP staining of control and rescued ES cells are shown (right).

Article Snippet: Polyclonal antibodies against ABCF1 (13950-1-AP) were purchased from ProteinTech; XPC (A301-122A) and mouse Nanog (A300-397A) from Bethyl Laboratories; DKC1 (H-300), OCT4 (N-19), and Pol II (N-20) from Santa Cruz Biotechnology; SOX2 (AB5603) from EMD Millipore; V5 ChIP grade (ab15828) from Abcam; and histone H3 (ChIP formulated, no. 2650) from Cell Signaling Technology (CST).

Techniques: Incubation, Plasmid Preparation, Expressing, Immunoprecipitation, Western Blot, Rescue Assay, Staining

Journal: Science Advances

Article Title: ATP-binding cassette protein ABCF1 couples transcription and genome surveillance in embryonic stem cells through low-complexity domain

doi: 10.1126/sciadv.abk2775

Figure Lengend Snippet: ( A ) WCEs from D3 mouse ES cells stably expressing V5-tagged FL ABCF1 or NBDs are incubated with three different 5′ biotinylated 98-mer oligonucleotides: single-stranded (ss), double-stranded (ds) with SOX2-binding motif (matched, ds-M), or ds lacking the sox-motif (unmatched, ds-UM). ( B ) Representative images of droplet formation with FL ABCF1 (9 μM) in the presence of 12.5 nM of Cy5-labeled (magenta) ss or ds-UM DNAs. Number and size of droplets are analyzed. Scale bar, 20 μm. ( C ) Phase separation diagram of FL ABCF1 with or without ds-UM at indicated concentrations. Blue and red dots indicate the presence and absence of droplets, respectively. Bottom: Fluorescence microscopy images showing that dsDNA (ds-UM) stimulates droplet formation of FL ABCF1 at low protein concentration (0.025 μM). ( D ) DNA copurified with ABCF1 IP from WCEs prepared from ETO-treated (20 μM) V5-ABCF1 knock-in mouse ES cells, denoted by vertical bar. ( E ) The percentage of stable ES cells (GFP, FL, or NBDs) containing puncta per image field upon treatment with DMSO or ETO (2 and 20 μM) is analyzed ( n = 9 to 10). Representative images of cells expressing GFP, FL, or NBDs treated with ETO (20 μM) are indicated (right). Scale bar, 20 μm.

Article Snippet: Polyclonal antibodies against ABCF1 (13950-1-AP) were purchased from ProteinTech; XPC (A301-122A) and mouse Nanog (A300-397A) from Bethyl Laboratories; DKC1 (H-300), OCT4 (N-19), and Pol II (N-20) from Santa Cruz Biotechnology; SOX2 (AB5603) from EMD Millipore; V5 ChIP grade (ab15828) from Abcam; and histone H3 (ChIP formulated, no. 2650) from Cell Signaling Technology (CST).

Techniques: Stable Transfection, Expressing, Incubation, Binding Assay, Labeling, Fluorescence, Microscopy, Protein Concentration, Knock-In

Journal: Science Advances

Article Title: ATP-binding cassette protein ABCF1 couples transcription and genome surveillance in embryonic stem cells through low-complexity domain

doi: 10.1126/sciadv.abk2775

Figure Lengend Snippet: ( A ) dsDNA transfection (left) or ETO treatment (20 μM; right) disrupts ABCF1-SOX2 interaction. Input (IN) and IgG or SOX2 IPs from WCEs of V5-ABCF1 knock-in mouse ES cells are analyzed by Western blotting. ( B ) Mouse ES cells transfected with 5′ 6-carboxyfluorescein (6-FAM)–labeled ss, ds-M, or ds-UM are enriched by FACS. Expression levels of pluripotency are analyzed by qPCR. ( C ) Representative images of droplet formation with FL ABCF1-GFP (green) and SOX2-mCherry (red) in the presence or absence of Cy5-labeled dsDNA (ds-UM, magenta). Scale bars, 20 μm. Fluorescence intensity levels of FL and SOX2 that colocalize in droplets are obtained as described in Materials and Methods. Bottom: Relative fluorescence intensity and size of SOX2 signals that overlap with FL in droplets are quantified from independent images ( n = 5). ( D ) Line-scan profiles of fluorescence intensity of FL, SOX2, and dsDNA in indicated droplets. ( E ) MNase-ChIP of ABCF1 in DMSO- and ETO-treated V5-ABCF1 knock-in mouse ES cells. Enrichment of ABCF1 is analyzed by qPCR as in . ( F ) MNase-ChIP of ABCF1 in DMSO- and 1,6-Hex (1.5%)–treated V5-ABCF1 knock-in mouse ES cells. Enrichment of ABCF1 (left) and Pol II (right) is analyzed by qPCR as in (E). ( G ) Colony formation assays in control and ABCF1 gain-of-function D3 mouse ES cells. AP-positive colonies are counted and indicated as a relative unit. Representative images of each AP-stained cells are indicated (right).

Article Snippet: Polyclonal antibodies against ABCF1 (13950-1-AP) were purchased from ProteinTech; XPC (A301-122A) and mouse Nanog (A300-397A) from Bethyl Laboratories; DKC1 (H-300), OCT4 (N-19), and Pol II (N-20) from Santa Cruz Biotechnology; SOX2 (AB5603) from EMD Millipore; V5 ChIP grade (ab15828) from Abcam; and histone H3 (ChIP formulated, no. 2650) from Cell Signaling Technology (CST).

Techniques: Transfection, Knock-In, Western Blot, Labeling, Expressing, Fluorescence, Staining

Journal: Science Advances

Article Title: ATP-binding cassette protein ABCF1 couples transcription and genome surveillance in embryonic stem cells through low-complexity domain

doi: 10.1126/sciadv.abk2775

Figure Lengend Snippet: ( A ) Chromatography scheme for purification of SCC-B from NT2 nuclear extracts (NT2 NE). NT2 NE is first subjected to ammonium sulfate precipitation (55% saturation) followed by a series of chromatographic columns including nickel affinity agarose (Ni-NTA), cation exchangers phosphocellulose (P11), heparin (Poros-HE), Mono S, anion exchanger Poros-HQ, hydroxyapatite (HAP), and gel filtration medium Superose 6. ( B ) Input fraction containing SCC-B activity from the Poros-HE step (IN), flow-through (FT), and various salt-eluted Mono S fractions was assayed for their ability to stimulate OCT4/SOX2-dependent transcription from the human NANOG promoter template engineered with four extra copies of the oct-sox composite binding element (bottom). All reactions contain purified general transcription factors (GTFs), Pol II, OCT4, SOX2, and recombinant XPC and DKC1 complexes. Transcribed RNA products are subjected to primer extension and visualized by autoradiography. ( C ) Mono S fractions assayed in in vitro transcription are separated on a polyacrylamide gel and stained with silver. Filled arrowhead indicates the ~110-kDa polypeptide that comigrates with SCC-B transcriptional activity. ( D ) Coomassie staining of Mono S fraction 14 demonstrates purification to homogeneity. ( E ) ABCF1 is enriched in human ES cells. Down-regulation of ABCF1 in human ES cell line H9 upon exit from pluripotency. ( F ) ABCF1 is enriched in mouse ES cells. mRNA and protein levels of ABCF1 in pluripotent D3 mouse ES cells carrying V5 epitope–tagged Abcf1 alleles (V5-ABCF1 knock-in) are compared to their differentiated counterparts.

Article Snippet: Polyclonal antibodies against ABCF1 (13950-1-AP) were purchased from ProteinTech; XPC (A301-122A) and mouse Nanog (A300-397A) from Bethyl Laboratories; DKC1 (H-300), OCT4 (N-19), and Pol II (N-20) from Santa Cruz Biotechnology; SOX2 (AB5603) from EMD Millipore; V5 ChIP grade (ab15828) from Abcam; and histone H3 (ChIP formulated, no. 2650) from Cell Signaling Technology (CST).

Techniques: Chromatography, Purification, Filtration, Activity Assay, Binding Assay, Recombinant, Autoradiography, In Vitro, Staining, Knock-In

Journal: Science Advances

Article Title: ATP-binding cassette protein ABCF1 couples transcription and genome surveillance in embryonic stem cells through low-complexity domain

doi: 10.1126/sciadv.abk2775

Figure Lengend Snippet: ( A ) Schematic diagram of FL wild-type (WT) ABCF1 protein depicting the N-terminal LCD (yellow) containing a polyglutamine (polyQ) tract and lysine (K)/glutamic acid (E)–rich regions. The two conserved lysine residues (K324 and K664) in the Walker A motif of each of the two NBDs (blue) in ABCF1 are highlighted. FL WT ABCF1, ATP-binding defective lysine-to-methionine mutant (2KM), various truncated ABCF1 proteins lacking part (Δ248 and Δ115), or all of the LCD (Δ302) as well as the LCD by itself (1–302) are purified from E. coli . ( B ) Transcriptional activities of the various recombinant ABCF1 proteins shown in (A) are assayed (over a twofold concentration range) together with recombinant XPC and DKC1 complexes in in vitro transcription as described in . ( C ) Schematic representation of the human and mouse ABCF1 and yeast homolog GCN20. The percentage of sequence similarity among human, mouse, and yeast homolog is indicated. Domain-swapped hybrid protein between the human LCD and yeast NBDs (H/Y) is generated and purified from E. coli . ( D ) Titration over a twofold concentration range of human and mouse ABCF1, yeast GCN20, and human-yeast hybrid (H/Y) proteins is assayed in in vitro transcription reactions.

Article Snippet: Polyclonal antibodies against ABCF1 (13950-1-AP) were purchased from ProteinTech; XPC (A301-122A) and mouse Nanog (A300-397A) from Bethyl Laboratories; DKC1 (H-300), OCT4 (N-19), and Pol II (N-20) from Santa Cruz Biotechnology; SOX2 (AB5603) from EMD Millipore; V5 ChIP grade (ab15828) from Abcam; and histone H3 (ChIP formulated, no. 2650) from Cell Signaling Technology (CST).

Techniques: Binding Assay, Mutagenesis, Purification, Recombinant, Concentration Assay, In Vitro, Sequencing, Generated, Titration

Journal: Science Advances

Article Title: ATP-binding cassette protein ABCF1 couples transcription and genome surveillance in embryonic stem cells through low-complexity domain

doi: 10.1126/sciadv.abk2775

Figure Lengend Snippet: ( A ) Chromatography scheme for purification of SCC-B from NT2 nuclear extracts (NT2 NE). NT2 NE is first subjected to ammonium sulfate precipitation (55% saturation) followed by a series of chromatographic columns including nickel affinity agarose (Ni-NTA), cation exchangers phosphocellulose (P11), heparin (Poros-HE), Mono S, anion exchanger Poros-HQ, hydroxyapatite (HAP), and gel filtration medium Superose 6. ( B ) Input fraction containing SCC-B activity from the Poros-HE step (IN), flow-through (FT), and various salt-eluted Mono S fractions was assayed for their ability to stimulate OCT4/SOX2-dependent transcription from the human NANOG promoter template engineered with four extra copies of the oct-sox composite binding element (bottom). All reactions contain purified general transcription factors (GTFs), Pol II, OCT4, SOX2, and recombinant XPC and DKC1 complexes. Transcribed RNA products are subjected to primer extension and visualized by autoradiography. ( C ) Mono S fractions assayed in in vitro transcription are separated on a polyacrylamide gel and stained with silver. Filled arrowhead indicates the ~110-kDa polypeptide that comigrates with SCC-B transcriptional activity. ( D ) Coomassie staining of Mono S fraction 14 demonstrates purification to homogeneity. ( E ) ABCF1 is enriched in human ES cells. Down-regulation of ABCF1 in human ES cell line H9 upon exit from pluripotency. ( F ) ABCF1 is enriched in mouse ES cells. mRNA and protein levels of ABCF1 in pluripotent D3 mouse ES cells carrying V5 epitope–tagged Abcf1 alleles (V5-ABCF1 knock-in) are compared to their differentiated counterparts.

Article Snippet: Polyclonal antibodies against ABCF1 (13950-1-AP) were purchased from ProteinTech; XPC (A301-122A) and mouse Nanog (A300-397A) from Bethyl Laboratories; DKC1 (H-300), OCT4 (N-19), and Pol II (N-20) from Santa Cruz Biotechnology; SOX2 (AB5603) from EMD Millipore; V5 ChIP grade (ab15828) from Abcam; and histone H3 (ChIP formulated, no. 2650) from Cell Signaling Technology (CST).

Techniques: Chromatography, Purification, Filtration, Activity Assay, Binding Assay, Recombinant, Autoradiography, In Vitro, Staining, Knock-In

Journal: Science Advances

Article Title: ATP-binding cassette protein ABCF1 couples transcription and genome surveillance in embryonic stem cells through low-complexity domain

doi: 10.1126/sciadv.abk2775

Figure Lengend Snippet: ( A ) shRNA-mediated knockdown of ABCF1 in mouse ES cells. Asterisk denotes a nonspecific band. ( B ) Depletion of ABCF1 in mouse ES cells leads to colony collapse with flattened cell morphology and reduced alkaline phosphatase (AP) staining, indicating spontaneous differentiation. ( C ) Loss of ABCF1 in mouse ES cells compromises pluripotency gene expression. Quantification of mRNA levels of pluripotency genes is analyzed by qPCR and normalized to Actb . ( D ) Depletion of ABCF1 induces expression of genes associated with the three embryonic germ layers and the trophectoderm, analyzed by qPCR as in (C). ( E ) Depletion of ABCF1 blocks somatic cell reprogramming. AP + colonies are counted after 14 days (11 days with doxycycline followed by 3 days of dox withdrawal) post-induction (dpi). ( F ) Single-cell suspensions of 14 dpi reprogrammed CF-1 MEFs as described in (E) are stained with anti-mouse SSEA-1 and analyzed by flow cytometry. ( G ) Micrococcal nuclease (MNase) ChIP analysis of ABCF1 occupancy on control and enhancer regions of Oct4 , Sox2 , and Nanog gene loci in V5-ABCF1 knock-in D3 mouse ES cells. Representative data showing the enrichment of V5-ABCF1 (gray bars) compared to control IgGs (white bars) are analyzed by qPCR and expressed as percentage of input chromatin. Schematic diagrams of OCT4/SOX2 binding sites of each gene and the relative positions of the amplicons are shown at the bottom. ( H ) ABCF1 is not recruited to the promoter of housekeeping gene Actb .

Article Snippet: Polyclonal antibodies against ABCF1 (13950-1-AP) were purchased from ProteinTech; XPC (A301-122A) and mouse Nanog (A300-397A) from Bethyl Laboratories; DKC1 (H-300), OCT4 (N-19), and Pol II (N-20) from Santa Cruz Biotechnology; SOX2 (AB5603) from EMD Millipore; V5 ChIP grade (ab15828) from Abcam; and histone H3 (ChIP formulated, no. 2650) from Cell Signaling Technology (CST).

Techniques: shRNA, Knockdown, Staining, Gene Expression, Expressing, Flow Cytometry, Control, Knock-In, Binding Assay

Journal: Science Advances

Article Title: ATP-binding cassette protein ABCF1 couples transcription and genome surveillance in embryonic stem cells through low-complexity domain

doi: 10.1126/sciadv.abk2775

Figure Lengend Snippet: ( A ) Top: Unstructured regions in human ABCF1. X axis indicates position of the amino acids, and Y axis shows the probability of disordered sequences. Regions that are above the value of 0.5 are predicted to be unstructured. The schematic diagram denotes protein domains of ABCF1: intrinsically disordered low-complexity domain (LCD; yellow, amino acids 1 to 302) and two nucleotide-binding domains (NBDs; blue). Bottom: Amino acid composition of human ABCF1. Each of the 20 amino acids is counted and marked as a black bar at that position in ABCF1. One-letter abbreviations for amino acids are used. Q, E, and K residues in the LCD are highlighted. ( B ) Schematic diagrams of recombinant GFP fusion ABCF1 proteins used in vitro droplet assays. ( C ) Representative images of droplet formation with FL ABCF1, NBDs, LCD, or GFP. Proteins (13 μM) are added to droplet formation buffer containing 200 mM NaCl. The average number and size (arbitrary unit) of droplets in each image are indicated ( n = 10). ( D ) Representative images of droplet formation with FL ( n = 10) and LCD ( n = 5) in the presence of buffer (Ctrl) or 10% 1,6-hexanediol (1,6-Hex). Relative droplet size of FL and LCD is indicated. ( E ) Fluorescence images of stable mouse D3 ES cell lines expressing GFP, FL ABCF1, or NBDs. Scale bars, 20 μm. ( F ) The percentage of cells having nuclear puncta per image field in the presence of buffer (Ctrl) or 3% 1,6-Hex is analyzed ( n = 9 to 10).

Article Snippet: Polyclonal antibodies against ABCF1 (13950-1-AP) were purchased from ProteinTech; XPC (A301-122A) and mouse Nanog (A300-397A) from Bethyl Laboratories; DKC1 (H-300), OCT4 (N-19), and Pol II (N-20) from Santa Cruz Biotechnology; SOX2 (AB5603) from EMD Millipore; V5 ChIP grade (ab15828) from Abcam; and histone H3 (ChIP formulated, no. 2650) from Cell Signaling Technology (CST).

Techniques: Binding Assay, Recombinant, In Vitro, Fluorescence, Expressing

Journal: Science Advances

Article Title: ATP-binding cassette protein ABCF1 couples transcription and genome surveillance in embryonic stem cells through low-complexity domain

doi: 10.1126/sciadv.abk2775

Figure Lengend Snippet: ( A ) Schematic diagram of FL wild-type (WT) ABCF1 protein depicting the N-terminal LCD (yellow) containing a polyglutamine (polyQ) tract and lysine (K)/glutamic acid (E)–rich regions. The two conserved lysine residues (K324 and K664) in the Walker A motif of each of the two NBDs (blue) in ABCF1 are highlighted. FL WT ABCF1, ATP-binding defective lysine-to-methionine mutant (2KM), various truncated ABCF1 proteins lacking part (Δ248 and Δ115), or all of the LCD (Δ302) as well as the LCD by itself (1–302) are purified from E. coli . ( B ) Transcriptional activities of the various recombinant ABCF1 proteins shown in (A) are assayed (over a twofold concentration range) together with recombinant XPC and DKC1 complexes in in vitro transcription as described in . ( C ) Schematic representation of the human and mouse ABCF1 and yeast homolog GCN20. The percentage of sequence similarity among human, mouse, and yeast homolog is indicated. Domain-swapped hybrid protein between the human LCD and yeast NBDs (H/Y) is generated and purified from E. coli . ( D ) Titration over a twofold concentration range of human and mouse ABCF1, yeast GCN20, and human-yeast hybrid (H/Y) proteins is assayed in in vitro transcription reactions.

Article Snippet: Polyclonal antibodies against ABCF1 (13950-1-AP) were purchased from ProteinTech; XPC (A301-122A) and mouse Nanog (A300-397A) from Bethyl Laboratories; DKC1 (H-300), OCT4 (N-19), and Pol II (N-20) from Santa Cruz Biotechnology; SOX2 (AB5603) from EMD Millipore; V5 ChIP grade (ab15828) from Abcam; and histone H3 (ChIP formulated, no. 2650) from Cell Signaling Technology (CST).

Techniques: Binding Assay, Mutagenesis, Purification, Recombinant, Concentration Assay, In Vitro, Sequencing, Generated, Titration

Journal: Science Advances

Article Title: ATP-binding cassette protein ABCF1 couples transcription and genome surveillance in embryonic stem cells through low-complexity domain

doi: 10.1126/sciadv.abk2775

Figure Lengend Snippet: ( A ) GST fusion proteins containing the LCD of human ABCF1 (1–302), the N-terminal domain of yeast GCN20 (1–197), and the transactivation domain of human transcription factor SREBP1a (1–50) are incubated with buffer only (−) or NT2 NEs (+). Input (IN) indicated. ( B ) Whole-cell extracts (WCEs) from 293T cells cotransfected with plasmid expressing V5-tagged ABCF1 together with either empty plasmid (−) or plasmids expressing FLAG-tagged SOX2 (S) or OCT4 (O) are immunoprecipitated with anti-FLAG antibody. ( C ) Input V5-ABCF1 KI mouse ES cell WCEs (IN) and IPs by IgG and anti-SOX2 antibodies are analyzed by Western blotting. ( D ) HA IPs from 293T cells overexpressing HA-tagged SOX2 (SOX2-HA) with V5-tagged FL or LCD-truncated human ABCF1 (NBDs). ( E ) ABCF1 knockdown rescue assay. mRNAs from control mouse ES cells (shNT) overexpressing RFP, and ABCF1 knockdown ES cells (sh1) overexpressing RFP, V5-tagged FL, LCD-truncated human ABCF1 (NBDs), or mouse NANOG are analyzed for Nanog , Fgf4 , and Klf4 mRNA levels by qPCR. ( F ) Colony formation assays of cells described in (E). Differentiation status is evaluated on the basis of AP staining intensity and colony morphology. Representative images of AP staining of control and rescued ES cells are shown (right).

Article Snippet: Polyclonal antibodies against ABCF1 (13950-1-AP) were purchased from ProteinTech; XPC (A301-122A) and mouse Nanog (A300-397A) from Bethyl Laboratories; DKC1 (H-300), OCT4 (N-19), and Pol II (N-20) from Santa Cruz Biotechnology; SOX2 (AB5603) from EMD Millipore; V5 ChIP grade (ab15828) from Abcam; and histone H3 (ChIP formulated, no. 2650) from Cell Signaling Technology (CST).

Techniques: Incubation, Plasmid Preparation, Expressing, Immunoprecipitation, Western Blot, Knockdown, Rescue Assay, Control, Staining

Journal: Science Advances

Article Title: ATP-binding cassette protein ABCF1 couples transcription and genome surveillance in embryonic stem cells through low-complexity domain

doi: 10.1126/sciadv.abk2775

Figure Lengend Snippet: ( A ) WCEs from D3 mouse ES cells stably expressing V5-tagged FL ABCF1 or NBDs are incubated with three different 5′ biotinylated 98-mer oligonucleotides: single-stranded (ss), double-stranded (ds) with SOX2-binding motif (matched, ds-M), or ds lacking the sox-motif (unmatched, ds-UM). ( B ) Representative images of droplet formation with FL ABCF1 (9 μM) in the presence of 12.5 nM of Cy5-labeled (magenta) ss or ds-UM DNAs. Number and size of droplets are analyzed. Scale bar, 20 μm. ( C ) Phase separation diagram of FL ABCF1 with or without ds-UM at indicated concentrations. Blue and red dots indicate the presence and absence of droplets, respectively. Bottom: Fluorescence microscopy images showing that dsDNA (ds-UM) stimulates droplet formation of FL ABCF1 at low protein concentration (0.025 μM). ( D ) DNA copurified with ABCF1 IP from WCEs prepared from ETO-treated (20 μM) V5-ABCF1 knock-in mouse ES cells, denoted by vertical bar. ( E ) The percentage of stable ES cells (GFP, FL, or NBDs) containing puncta per image field upon treatment with DMSO or ETO (2 and 20 μM) is analyzed ( n = 9 to 10). Representative images of cells expressing GFP, FL, or NBDs treated with ETO (20 μM) are indicated (right). Scale bar, 20 μm.

Article Snippet: Polyclonal antibodies against ABCF1 (13950-1-AP) were purchased from ProteinTech; XPC (A301-122A) and mouse Nanog (A300-397A) from Bethyl Laboratories; DKC1 (H-300), OCT4 (N-19), and Pol II (N-20) from Santa Cruz Biotechnology; SOX2 (AB5603) from EMD Millipore; V5 ChIP grade (ab15828) from Abcam; and histone H3 (ChIP formulated, no. 2650) from Cell Signaling Technology (CST).

Techniques: Stable Transfection, Expressing, Incubation, Binding Assay, Labeling, Fluorescence, Microscopy, Protein Concentration, Knock-In

Journal: Science Advances

Article Title: ATP-binding cassette protein ABCF1 couples transcription and genome surveillance in embryonic stem cells through low-complexity domain

doi: 10.1126/sciadv.abk2775

Figure Lengend Snippet: ( A ) dsDNA transfection (left) or ETO treatment (20 μM; right) disrupts ABCF1-SOX2 interaction. Input (IN) and IgG or SOX2 IPs from WCEs of V5-ABCF1 knock-in mouse ES cells are analyzed by Western blotting. ( B ) Mouse ES cells transfected with 5′ 6-carboxyfluorescein (6-FAM)–labeled ss, ds-M, or ds-UM are enriched by FACS. Expression levels of pluripotency are analyzed by qPCR. ( C ) Representative images of droplet formation with FL ABCF1-GFP (green) and SOX2-mCherry (red) in the presence or absence of Cy5-labeled dsDNA (ds-UM, magenta). Scale bars, 20 μm. Fluorescence intensity levels of FL and SOX2 that colocalize in droplets are obtained as described in Materials and Methods. Bottom: Relative fluorescence intensity and size of SOX2 signals that overlap with FL in droplets are quantified from independent images ( n = 5). ( D ) Line-scan profiles of fluorescence intensity of FL, SOX2, and dsDNA in indicated droplets. ( E ) MNase-ChIP of ABCF1 in DMSO- and ETO-treated V5-ABCF1 knock-in mouse ES cells. Enrichment of ABCF1 is analyzed by qPCR as in . ( F ) MNase-ChIP of ABCF1 in DMSO- and 1,6-Hex (1.5%)–treated V5-ABCF1 knock-in mouse ES cells. Enrichment of ABCF1 (left) and Pol II (right) is analyzed by qPCR as in (E). ( G ) Colony formation assays in control and ABCF1 gain-of-function D3 mouse ES cells. AP-positive colonies are counted and indicated as a relative unit. Representative images of each AP-stained cells are indicated (right).

Article Snippet: Polyclonal antibodies against ABCF1 (13950-1-AP) were purchased from ProteinTech; XPC (A301-122A) and mouse Nanog (A300-397A) from Bethyl Laboratories; DKC1 (H-300), OCT4 (N-19), and Pol II (N-20) from Santa Cruz Biotechnology; SOX2 (AB5603) from EMD Millipore; V5 ChIP grade (ab15828) from Abcam; and histone H3 (ChIP formulated, no. 2650) from Cell Signaling Technology (CST).

Techniques: Transfection, Knock-In, Western Blot, Labeling, Expressing, Fluorescence, Control, Staining

Journal: Science Advances

Article Title: ATP-binding cassette protein ABCF1 couples transcription and genome surveillance in embryonic stem cells through low-complexity domain

doi: 10.1126/sciadv.abk2775

Figure Lengend Snippet: ( A ) Chromatography scheme for purification of SCC-B from NT2 nuclear extracts (NT2 NE). NT2 NE is first subjected to ammonium sulfate precipitation (55% saturation) followed by a series of chromatographic columns including nickel affinity agarose (Ni-NTA), cation exchangers phosphocellulose (P11), heparin (Poros-HE), Mono S, anion exchanger Poros-HQ, hydroxyapatite (HAP), and gel filtration medium Superose 6. ( B ) Input fraction containing SCC-B activity from the Poros-HE step (IN), flow-through (FT), and various salt-eluted Mono S fractions was assayed for their ability to stimulate OCT4/SOX2-dependent transcription from the human NANOG promoter template engineered with four extra copies of the oct-sox composite binding element (bottom). All reactions contain purified general transcription factors (GTFs), Pol II, OCT4, SOX2, and recombinant XPC and DKC1 complexes. Transcribed RNA products are subjected to primer extension and visualized by autoradiography. ( C ) Mono S fractions assayed in in vitro transcription are separated on a polyacrylamide gel and stained with silver. Filled arrowhead indicates the ~110-kDa polypeptide that comigrates with SCC-B transcriptional activity. ( D ) Coomassie staining of Mono S fraction 14 demonstrates purification to homogeneity. ( E ) ABCF1 is enriched in human ES cells. Down-regulation of ABCF1 in human ES cell line H9 upon exit from pluripotency. ( F ) ABCF1 is enriched in mouse ES cells. mRNA and protein levels of ABCF1 in pluripotent D3 mouse ES cells carrying V5 epitope–tagged Abcf1 alleles (V5-ABCF1 knock-in) are compared to their differentiated counterparts.

Article Snippet: Polyclonal antibodies against ABCF1 (13950-1-AP) were purchased from ProteinTech; XPC (A301-122A) and mouse Nanog (A300-397A) from Bethyl Laboratories; DKC1 (H-300), OCT4 (N-19), and Pol II (N-20) from Santa Cruz Biotechnology; SOX2 (AB5603) from EMD Millipore; V5 ChIP grade (ab15828) from Abcam; and histone H3 (ChIP formulated, no. 2650) from Cell Signaling Technology (CST).

Techniques: Chromatography, Purification, Filtration, Activity Assay, Binding Assay, Recombinant, Autoradiography, In Vitro, Staining, Knock-In

Journal: Science Advances

Article Title: ATP-binding cassette protein ABCF1 couples transcription and genome surveillance in embryonic stem cells through low-complexity domain

doi: 10.1126/sciadv.abk2775

Figure Lengend Snippet: ( A ) Schematic diagram of FL wild-type (WT) ABCF1 protein depicting the N-terminal LCD (yellow) containing a polyglutamine (polyQ) tract and lysine (K)/glutamic acid (E)–rich regions. The two conserved lysine residues (K324 and K664) in the Walker A motif of each of the two NBDs (blue) in ABCF1 are highlighted. FL WT ABCF1, ATP-binding defective lysine-to-methionine mutant (2KM), various truncated ABCF1 proteins lacking part (Δ248 and Δ115), or all of the LCD (Δ302) as well as the LCD by itself (1–302) are purified from E. coli . ( B ) Transcriptional activities of the various recombinant ABCF1 proteins shown in (A) are assayed (over a twofold concentration range) together with recombinant XPC and DKC1 complexes in in vitro transcription as described in . ( C ) Schematic representation of the human and mouse ABCF1 and yeast homolog GCN20. The percentage of sequence similarity among human, mouse, and yeast homolog is indicated. Domain-swapped hybrid protein between the human LCD and yeast NBDs (H/Y) is generated and purified from E. coli . ( D ) Titration over a twofold concentration range of human and mouse ABCF1, yeast GCN20, and human-yeast hybrid (H/Y) proteins is assayed in in vitro transcription reactions.

Article Snippet: Polyclonal antibodies against ABCF1 (13950-1-AP) were purchased from ProteinTech; XPC (A301-122A) and mouse Nanog (A300-397A) from Bethyl Laboratories; DKC1 (H-300), OCT4 (N-19), and Pol II (N-20) from Santa Cruz Biotechnology; SOX2 (AB5603) from EMD Millipore; V5 ChIP grade (ab15828) from Abcam; and histone H3 (ChIP formulated, no. 2650) from Cell Signaling Technology (CST).

Techniques: Binding Assay, Mutagenesis, Purification, Recombinant, Concentration Assay, In Vitro, Sequencing, Generated, Titration