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  • 94
    Thermo Fisher pcna
    CD24+ Ptch1 lox/lox ;GFAP cre cells co-label with markers of neural progenitors and granule cell precursors. (A-R) Co-immunolabelling of cytospun flow-sorted CD24+ and CD24- Ptch1 lox/lox ;GFAP cre primary cells with markers of progenitor and differentiated cells. Adjacent bar graphs illustrate the percentage of CD24+ (white bars) and CD24- (black bars) cells that co-labelled with their respective secondary marker. Co-labelled antibodies were <t>PCNA:</t> proliferating cells (A-C), Sox2: stem/progenitor cells (D-F), <t>NeuN:</t> mature neurons (G-I), βIIItubulin: neural progenitors (J-L), Pax6: radial glial progenitors (M-O) and BLBP: radial glial cells (P-R). Inset images show magnified images of the co-staining. Scale bar A, H, J, K and P 200μm, scale bar B, D, E, G, M, N and Q 100μm. NS: non-significant, *p
    Pcna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 836 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti pcna
    CD24+ Ptch1 lox/lox ;GFAP cre cells co-label with markers of neural progenitors and granule cell precursors. (A-R) Co-immunolabelling of cytospun flow-sorted CD24+ and CD24- Ptch1 lox/lox ;GFAP cre primary cells with markers of progenitor and differentiated cells. Adjacent bar graphs illustrate the percentage of CD24+ (white bars) and CD24- (black bars) cells that co-labelled with their respective secondary marker. Co-labelled antibodies were <t>PCNA:</t> proliferating cells (A-C), Sox2: stem/progenitor cells (D-F), <t>NeuN:</t> mature neurons (G-I), βIIItubulin: neural progenitors (J-L), Pax6: radial glial progenitors (M-O) and BLBP: radial glial cells (P-R). Inset images show magnified images of the co-staining. Scale bar A, H, J, K and P 200μm, scale bar B, D, E, G, M, N and Q 100μm. NS: non-significant, *p
    Anti Pcna, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 627 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Santa Cruz Biotechnology pcna
    Phosphorylation of transiently expressed OSU NSP1. (A) Alignment of C-terminal NSP1 sequences and a portion of IκB containing the DSGΦXS phosphodegron. Red lettering indicates OSU-specific residues. The phosphodegron region is boxed. (B) HEK293T cells were transfected with NSP1 expression vectors or maintained in the presence of MG132 with or without TNF-α, beginning at 20 h p.t. Lysates prepared from cells at 24 h p.t. were resolved by gel electrophoresis, in duplicate, and blotted onto nitrocellulose membranes. Membranes were mock treated (-CIP) or treated with CIP in parallel. Mock- and CIP-treated membranes were probed with <t>PCNA</t> antibody, p-IκB antibody to detect phosphorylated NSP1 (p-NSP1) and IκB (p-IκB), OSU NSP1 antibody to detect OSU and 4F-OSU NSP1, and <t>SA11-5S</t> antibody to detect SA11-4F and 4F-OSU NSP1. (C) Lysates prepared from HEK293T cells transfected with expression vectors for OSU NSP1 (OSU) or HALO-tagged OSU NSP1 (H-OSU) were resolved by gel electrophoresis and blotted onto nitrocellulose membranes, in duplicate. Mock- and CIP-treated membranes were probed with p-IκB antibody to detect p-NSP1 and with antibodies to the HALO tag and PCNA. Levels of phosphorylated H-OSU were calculated relative to PCNA levels and normalized to 100% for the untreated sample. Data shown are from two independent experiments (means ± standard deviations). *, P
    Pcna, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 4936 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc pcna
    Phosphorylation of transiently expressed OSU NSP1. (A) Alignment of C-terminal NSP1 sequences and a portion of IκB containing the DSGΦXS phosphodegron. Red lettering indicates OSU-specific residues. The phosphodegron region is boxed. (B) HEK293T cells were transfected with NSP1 expression vectors or maintained in the presence of MG132 with or without TNF-α, beginning at 20 h p.t. Lysates prepared from cells at 24 h p.t. were resolved by gel electrophoresis, in duplicate, and blotted onto nitrocellulose membranes. Membranes were mock treated (-CIP) or treated with CIP in parallel. Mock- and CIP-treated membranes were probed with <t>PCNA</t> antibody, p-IκB antibody to detect phosphorylated NSP1 (p-NSP1) and IκB (p-IκB), OSU NSP1 antibody to detect OSU and 4F-OSU NSP1, and <t>SA11-5S</t> antibody to detect SA11-4F and 4F-OSU NSP1. (C) Lysates prepared from HEK293T cells transfected with expression vectors for OSU NSP1 (OSU) or HALO-tagged OSU NSP1 (H-OSU) were resolved by gel electrophoresis and blotted onto nitrocellulose membranes, in duplicate. Mock- and CIP-treated membranes were probed with p-IκB antibody to detect p-NSP1 and with antibodies to the HALO tag and PCNA. Levels of phosphorylated H-OSU were calculated relative to PCNA levels and normalized to 100% for the untreated sample. Data shown are from two independent experiments (means ± standard deviations). *, P
    Pcna, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 2225 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology anti pcna
    <t>Polη</t> promotion of <t>PCNA</t> ubiquitination depends on PIP3 and PIP2, but not on PIP1. (A) Schematic structure of human Polη. Parts of PIP and UBZ sequences are shown. Amino-acid residues indicated by asterisks were replaced with alanines in the mutants. (B) Western blot analysis of FLAG-Polη-expressing cells. XP-V cells were transfected with the indicated plasmids for expression of FLAG-Polη (wt) or the indicated pip mutants, incubated for 24 h, irradiated with UV (15 J/m 2 ) and further incubated for 3 h. Whole-cell lysates (WCL) or chromatin fractions were subjected to western blotting with anti-PCNA, anti-Polη and anti-Lamin B (loading control) antibodies.
    Anti Pcna, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 2456 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam proliferating cell nuclear antigen
    <t>Polη</t> promotion of <t>PCNA</t> ubiquitination depends on PIP3 and PIP2, but not on PIP1. (A) Schematic structure of human Polη. Parts of PIP and UBZ sequences are shown. Amino-acid residues indicated by asterisks were replaced with alanines in the mutants. (B) Western blot analysis of FLAG-Polη-expressing cells. XP-V cells were transfected with the indicated plasmids for expression of FLAG-Polη (wt) or the indicated pip mutants, incubated for 24 h, irradiated with UV (15 J/m 2 ) and further incubated for 3 h. Whole-cell lysates (WCL) or chromatin fractions were subjected to western blotting with anti-PCNA, anti-Polη and anti-Lamin B (loading control) antibodies.
    Proliferating Cell Nuclear Antigen, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology cell nuclear antigen pcna
    The telencephalon neurogenic zone contains three cell states. Telencephalic cross sections at the anteroposterior level indicated in the inset on the bottom left in h . a–d , Single optical section in a gfap:GFP transgenic brain section, immunostained for GFP (green), <t>S100β</t> (red), and <t>PCNA</t> (blue), depicting a close-up view on the ventricular surface at the midline between the two brain hemispheres. gfap: GFP and S100β are coexpressed in all radial glial cells (yellow in c ). Three cell states are visible: the majority of radial glial cells are noncycling (PCNA-negative; green arrow, state I cells). Some radial glial cells are PCNA positive (yellow arrow, state II cells). Some cells express only PCNA and no radial glial marker (red arrow, state III cells). e–g , State III cells express markers of neuroblasts. e , f , Expression of ascl1a revealed by in situ hybridization (black signal), revealing colocalization with PCNA (red). g , PSA-NCAM antibody staining (green) labels many state III cells, as shown by red arrows, revealing their identity of migrating neuroblasts. h , Overview of a gfap:GFP telencephalic section in a confocal stack projection, stained for GFP (green) and PCNA (blue). The arrows depict the telencephalic ventricle, located at the surface and at the midline. The boxed area depicts the region shown in a–g . The white line depicts the pallial–subpallial border region, in which mostly state III cells are located. The white dots depict the pallial ventricular region in which states I, II, and III are intermingled. This latter region is quantified in i and examined further in the next experiments. i , Relative proportions of state I, II, and III cells, counted in 2845 VZ cells of four brains. Scale bars: (in d ) a–d , 10 μm; (in g ) e–g , 10 μm; h , 100 μm.
    Cell Nuclear Antigen Pcna, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 1455 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Agilent technologies pcna
    Immunohistochemical expression of <t>p53</t> (A) and <t>PCNA</t> (B) in calcifying cystic odontogenic tumor (400X).
    Pcna, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 94/100, based on 1155 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti pcna antibody
    Immunohistochemical expression of <t>p53</t> (A) and <t>PCNA</t> (B) in calcifying cystic odontogenic tumor (400X).
    Anti Pcna Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 481 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti pcna
    Immunohistochemical expression of <t>p53</t> (A) and <t>PCNA</t> (B) in calcifying cystic odontogenic tumor (400X).
    Anti Pcna, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 563 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Agilent technologies anti pcna
    Ligase and Fen1 have a shorter residence time at RF than <t>PCNA</t> ( A ) Half of the nucleus (BA) was bleached by one single, 12 s bleach pulse in live cells expressing <t>GFP-Ligase</t> or GFP-Fen1 and RFP-PCNA, resulting in an almost complete depletion of Ligase and Fen1 from the unbleached nucleoplasm and RF, in contrast to PCNA. ( B ) Different residence times of PCNA, Ligase and Fen1 at RF shown by loss of fluorescence from unbleached RF immediately after bleaching (16–20 RF from five cells were analyzed in each case). ( C ) Fast equilibration of the remaining Ligase and Fen1 between bleached and unbleached half of the nucleus within a few seconds in contrast to a slow equilibration of PCNA over minutes (three cells each). Scale bar, 5 μm.
    Anti Pcna, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 92/100, based on 564 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology mouse anti pcna
    MutLα interacts with <t>PCNA</t> in solution. ( A and B ) Examples of equilibrium gel filtration profiles after injection of 10 μL 10 μM wild-type ( A ) or <t>PMS2-Q721A</t> MutLα ( B ) onto a Superdex 200 column equilibrated with 10 μM
    Mouse Anti Pcna, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 581 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Agilent technologies cell nuclear antigen pcna
    Effect of CXCR2 on hepatocyte proliferation and liver regeneration after I/R injury (A) and partial hepatectomy (B) was determined by <t>immunohistochemical</t> staining for proliferating cell nuclear antigen <t>(PCNA)</t> and quantitative analysis of PCNA labeling. (A) Hepatocyte proliferation in post-ischemic liver (upper panel) after I/R injury showed a significant increase in CXCR2−/− mice. Data are mean ± SEM with n = 3–14 per group. * P
    Cell Nuclear Antigen Pcna, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 94/100, based on 604 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher pcna staining kit
    Effect of CXCR2 on hepatocyte proliferation and liver regeneration after I/R injury (A) and partial hepatectomy (B) was determined by <t>immunohistochemical</t> staining for proliferating cell nuclear antigen <t>(PCNA)</t> and quantitative analysis of PCNA labeling. (A) Hepatocyte proliferation in post-ischemic liver (upper panel) after I/R injury showed a significant increase in CXCR2−/− mice. Data are mean ± SEM with n = 3–14 per group. * P
    Pcna Staining Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 577 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti pcna antibody epr3821
    Effect of CXCR2 on hepatocyte proliferation and liver regeneration after I/R injury (A) and partial hepatectomy (B) was determined by <t>immunohistochemical</t> staining for proliferating cell nuclear antigen <t>(PCNA)</t> and quantitative analysis of PCNA labeling. (A) Hepatocyte proliferation in post-ischemic liver (upper panel) after I/R injury showed a significant increase in CXCR2−/− mice. Data are mean ± SEM with n = 3–14 per group. * P
    Anti Pcna Antibody Epr3821, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 117 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Agilent technologies mouse anti pcna
    Conservation of the zebrafish id1-CRM2 core sequences and its function across evolution. (A) Sequence comparison of zebrafish id1-CRM2-core (Danio) with human (Homo) and mouse (Mus) orthologous sequences. Conserved nucleotides are indicated with an asterisk. Conserved motifs are outlined by yellow boxes comprising putative DNA recognition sequences for the transcription factors FoxA2, Smad (SBM1 and 2), CRE binding protein (CREB), Pknox and EGR1. Nucleotide sequences in green, red or blue correspond to previously identified sequences in the mouse id1 orthologue: Smad binding element (SBE) , a Smad 1/5 binding site and a binding site for an unknown binding protein, respectively. (B-I) Immunohistochemistry of telencephalic transverse sections with antibodies against <t>GFP</t> (B, F), S100ß (C, G) and <t>PCNA</t> (D, H) (merged panels: E, I). (B) Expression of GFP in RGCs at the telencephalic ventricular zone driven by the human id1 regulatory sequences in the zebrafish adult telencephalon. (F) Expression of the human Tg(Hsid1-CRM2) driven reporter construct is up-regulated upon stab wound injury. The injured telencephalic hemisphere is labeled with a white asterisk. (J) Quantification of PCNA and S100ß expression in GFP + cells in the Tg(Hsid1-CRM2) line. (K) Relative population size of type 1 and type 2 RGCs in the control and lesioned hemispheres. The proportion of GFP + /S100β + /PCNA - type 1 and GFP + /S100 + /PCNA + type 2 stem cells is not altered in the injured hemisphere relative to the control hemisphere of the telencephalon. (L, M) Quantification of GFP + cells upon injury. The number of GFP-expressing cells (L) and the intensity ratio between left and right hemispheres comparing undamaged hemisphere (control) and damaged telencephalic hemisphere (M) are both increased following injury. Bars: mean ± SD. Significance is indicated by asterisks: *, .01≤ p
    Mouse Anti Pcna, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 93/100, based on 367 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam mouse anti pcna
    Generation of a <t>PCNA</t> K164R mutant cell line in RPE-1 using CRISPR/Cas9 A) Schematic of the human PCNA indicating that exon 5 was targeted by CRISPR-Cas9. The K164R mutation was knocked-in utilizing a donor plasmid. B) Schematic of screening PCR and expected PCR product sizes after EcoRI restriction enzyme digestion. C) Representative genotyping PCR. Not targeted (wildtype; 1426 bp), monoallelic knock-in (KIN) ( PCNA KR/- 1E4; 1426 bp, 1168 bp, 258 bp), and biallelic KIN ( PCNA KR/KR 1E12, 2B10; 1168bp, 258 bp). D) Karyotyping analysis from RPE-1 wildtype, PCNA KR/KR (1E12, 2B10) and PCNA KR/- (1E4) cell lines. Blue indicates expec ted RPE-1 karyotype. Red indicates chromosomal abnormalities. E) Western <t>blot</t> analyses of whole cell extracts from wildtype RPE-1, PCNA K164R , and RAD18 -/- cells for MCM2 with α-Tubulin as the loading control. Quantification of MCM2 levels normalized to loading control.
    Mouse Anti Pcna, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 324 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Santa Cruz Biotechnology rabbit anti pcna
    Phospholipid synthesis activation and AP-1 content in NPcis mice brain as compared to C57BL/6J WT or <t>fos</t> (−/−) KO mice. ( A ) NPcis mice at 4 months of age showing clear PNS tumor burden were treated during 28 days with ASO, SO or vehicle dispensed by an osmotic pump into the caudate putamen (CP) (see Materials and Methods ). After treatment, animals were sacrificed and CP examined by WB for c-Fos and <t>PCNA</t> expression, the latter as an indication of the proliferative status of the tissue. The first two lanes correspond to CP from the left (non-treated) and right (ASO-treated) brain hemispheres from the same NPcis mouse. The 3rd and 4th lanes correspond to the CP from the right, SO-treated or vehicle-treated hemisphere from NPcis mice. The last lane corresponds to CP from a non-treated NPcis mouse. One experiment representative of two performed is shown. Tubulin was stained as a loading control. ( B ) Phospholipid synthesis was measured in vitro in CP from NPcis mice non-treated (1st column), or treated during 28 days as indicated in (A) with SO (2nd column) or from NPcis mice treated only in the right hemisphere with ASO (3rd column, NPcis+ASO) and compared to CP from the left hemisphere of the ASO-treated animals (4th column, NPcis –ASO) or to WT (5th column) or C57BL/6J fos (−/−) KO mice (last column), as indicated. Results are the mean ± SD of two independent experiments performed in triplicate; *p
    Rabbit Anti Pcna, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 91/100, based on 308 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    CD24+ Ptch1 lox/lox ;GFAP cre cells co-label with markers of neural progenitors and granule cell precursors. (A-R) Co-immunolabelling of cytospun flow-sorted CD24+ and CD24- Ptch1 lox/lox ;GFAP cre primary cells with markers of progenitor and differentiated cells. Adjacent bar graphs illustrate the percentage of CD24+ (white bars) and CD24- (black bars) cells that co-labelled with their respective secondary marker. Co-labelled antibodies were PCNA: proliferating cells (A-C), Sox2: stem/progenitor cells (D-F), NeuN: mature neurons (G-I), βIIItubulin: neural progenitors (J-L), Pax6: radial glial progenitors (M-O) and BLBP: radial glial cells (P-R). Inset images show magnified images of the co-staining. Scale bar A, H, J, K and P 200μm, scale bar B, D, E, G, M, N and Q 100μm. NS: non-significant, *p

    Journal: PLoS ONE

    Article Title: Identification of CD24 as a marker of Patched1 deleted medulloblastoma-initiating neural progenitor cells

    doi: 10.1371/journal.pone.0210665

    Figure Lengend Snippet: CD24+ Ptch1 lox/lox ;GFAP cre cells co-label with markers of neural progenitors and granule cell precursors. (A-R) Co-immunolabelling of cytospun flow-sorted CD24+ and CD24- Ptch1 lox/lox ;GFAP cre primary cells with markers of progenitor and differentiated cells. Adjacent bar graphs illustrate the percentage of CD24+ (white bars) and CD24- (black bars) cells that co-labelled with their respective secondary marker. Co-labelled antibodies were PCNA: proliferating cells (A-C), Sox2: stem/progenitor cells (D-F), NeuN: mature neurons (G-I), βIIItubulin: neural progenitors (J-L), Pax6: radial glial progenitors (M-O) and BLBP: radial glial cells (P-R). Inset images show magnified images of the co-staining. Scale bar A, H, J, K and P 200μm, scale bar B, D, E, G, M, N and Q 100μm. NS: non-significant, *p

    Article Snippet: For murine tissue immunohistochemistry, antibodies used were Olig2 (Millipore, Massachusetts, USA, AB9610), CNPase (Millipore, MAB326), PCNA (Zymed/Thermo-Fisher, 13–3900), Calbindin (Sigma, C9848), NeuN (Chemicon/Thermo-Fisher, MAB377), Pax6 (Covance, USA, PRB-278P), Sox2 (Merck, New Jersey, USA, AB5603), βIIItubulin (Promega, Wisconsin, USA, G712A), BLBP (Millipore, ABN14).

    Techniques: Flow Cytometry, Marker, Staining

    Phosphorylation of transiently expressed OSU NSP1. (A) Alignment of C-terminal NSP1 sequences and a portion of IκB containing the DSGΦXS phosphodegron. Red lettering indicates OSU-specific residues. The phosphodegron region is boxed. (B) HEK293T cells were transfected with NSP1 expression vectors or maintained in the presence of MG132 with or without TNF-α, beginning at 20 h p.t. Lysates prepared from cells at 24 h p.t. were resolved by gel electrophoresis, in duplicate, and blotted onto nitrocellulose membranes. Membranes were mock treated (-CIP) or treated with CIP in parallel. Mock- and CIP-treated membranes were probed with PCNA antibody, p-IκB antibody to detect phosphorylated NSP1 (p-NSP1) and IκB (p-IκB), OSU NSP1 antibody to detect OSU and 4F-OSU NSP1, and SA11-5S antibody to detect SA11-4F and 4F-OSU NSP1. (C) Lysates prepared from HEK293T cells transfected with expression vectors for OSU NSP1 (OSU) or HALO-tagged OSU NSP1 (H-OSU) were resolved by gel electrophoresis and blotted onto nitrocellulose membranes, in duplicate. Mock- and CIP-treated membranes were probed with p-IκB antibody to detect p-NSP1 and with antibodies to the HALO tag and PCNA. Levels of phosphorylated H-OSU were calculated relative to PCNA levels and normalized to 100% for the untreated sample. Data shown are from two independent experiments (means ± standard deviations). *, P

    Journal: mBio

    Article Title: Rotavirus NSP1 Requires Casein Kinase II-Mediated Phosphorylation for Hijacking of Cullin-RING Ligases

    doi: 10.1128/mBio.01213-17

    Figure Lengend Snippet: Phosphorylation of transiently expressed OSU NSP1. (A) Alignment of C-terminal NSP1 sequences and a portion of IκB containing the DSGΦXS phosphodegron. Red lettering indicates OSU-specific residues. The phosphodegron region is boxed. (B) HEK293T cells were transfected with NSP1 expression vectors or maintained in the presence of MG132 with or without TNF-α, beginning at 20 h p.t. Lysates prepared from cells at 24 h p.t. were resolved by gel electrophoresis, in duplicate, and blotted onto nitrocellulose membranes. Membranes were mock treated (-CIP) or treated with CIP in parallel. Mock- and CIP-treated membranes were probed with PCNA antibody, p-IκB antibody to detect phosphorylated NSP1 (p-NSP1) and IκB (p-IκB), OSU NSP1 antibody to detect OSU and 4F-OSU NSP1, and SA11-5S antibody to detect SA11-4F and 4F-OSU NSP1. (C) Lysates prepared from HEK293T cells transfected with expression vectors for OSU NSP1 (OSU) or HALO-tagged OSU NSP1 (H-OSU) were resolved by gel electrophoresis and blotted onto nitrocellulose membranes, in duplicate. Mock- and CIP-treated membranes were probed with p-IκB antibody to detect p-NSP1 and with antibodies to the HALO tag and PCNA. Levels of phosphorylated H-OSU were calculated relative to PCNA levels and normalized to 100% for the untreated sample. Data shown are from two independent experiments (means ± standard deviations). *, P

    Article Snippet: Rabbit polyclonal antibodies to simian SA11-5S and porcine OSU NSP1 proteins ( ) and PCNA (sc-7907; Santa Cruz Biotech [SCB]), rabbit monoclonal antibody to β-TrCP (11984S; Cell Signaling Technology, Inc. [CST]), and mouse monoclonal antibodies to IκB (4814; CST), p-IκB (9246; CST), and CKIIα (sc-373894; SCB) were used at a 1:5,000 dilution.

    Techniques: Transfection, Expressing, Nucleic Acid Electrophoresis

    Effect of priming loop mutations on NSP1 phosphorylation. (A) Lysates prepared from HEK293T cells expressing WT OSU NSP1 and forms of the protein with the indicated mutations were analyzed by immunoblot assay using p-IκB antibody to recognize p-NSP1, OSU NSP1 antibody, and PCNA antibody. (B) Predicted phosphorylation patterns of the OSU NSP1 ILD by CKI and CKII, using D477 and E486 as priming residues, respectively.

    Journal: mBio

    Article Title: Rotavirus NSP1 Requires Casein Kinase II-Mediated Phosphorylation for Hijacking of Cullin-RING Ligases

    doi: 10.1128/mBio.01213-17

    Figure Lengend Snippet: Effect of priming loop mutations on NSP1 phosphorylation. (A) Lysates prepared from HEK293T cells expressing WT OSU NSP1 and forms of the protein with the indicated mutations were analyzed by immunoblot assay using p-IκB antibody to recognize p-NSP1, OSU NSP1 antibody, and PCNA antibody. (B) Predicted phosphorylation patterns of the OSU NSP1 ILD by CKI and CKII, using D477 and E486 as priming residues, respectively.

    Article Snippet: Rabbit polyclonal antibodies to simian SA11-5S and porcine OSU NSP1 proteins ( ) and PCNA (sc-7907; Santa Cruz Biotech [SCB]), rabbit monoclonal antibody to β-TrCP (11984S; Cell Signaling Technology, Inc. [CST]), and mouse monoclonal antibodies to IκB (4814; CST), p-IκB (9246; CST), and CKIIα (sc-373894; SCB) were used at a 1:5,000 dilution.

    Techniques: Expressing

    Effect of NSP1 phosphorylation on interactions with β-TrCP. HEK293T cells were transfected with vectors expressing FLAG–β-TrCP and WT or mutant OSU NSP1. Lysates prepared from the cells at 24 h p.t. were incubated with anti-FLAG resin to immunoprecipitate complexes containing FLAG–β-TrCP. Input fractions and eluted proteins were analyzed by immunoblot assay with antibodies specific for NSP1, FLAG, p-NSP1, p-IκB, and PCNA. The OSU NSP1 antibody was generated using a peptide representing the C terminus of the OSU NSP1 proteins ( 4 ). As a result, the OSU NSP1 antibody was not able to recognize the product of the OSU ΔC13 expression vector ( 5 ).

    Journal: mBio

    Article Title: Rotavirus NSP1 Requires Casein Kinase II-Mediated Phosphorylation for Hijacking of Cullin-RING Ligases

    doi: 10.1128/mBio.01213-17

    Figure Lengend Snippet: Effect of NSP1 phosphorylation on interactions with β-TrCP. HEK293T cells were transfected with vectors expressing FLAG–β-TrCP and WT or mutant OSU NSP1. Lysates prepared from the cells at 24 h p.t. were incubated with anti-FLAG resin to immunoprecipitate complexes containing FLAG–β-TrCP. Input fractions and eluted proteins were analyzed by immunoblot assay with antibodies specific for NSP1, FLAG, p-NSP1, p-IκB, and PCNA. The OSU NSP1 antibody was generated using a peptide representing the C terminus of the OSU NSP1 proteins ( 4 ). As a result, the OSU NSP1 antibody was not able to recognize the product of the OSU ΔC13 expression vector ( 5 ).

    Article Snippet: Rabbit polyclonal antibodies to simian SA11-5S and porcine OSU NSP1 proteins ( ) and PCNA (sc-7907; Santa Cruz Biotech [SCB]), rabbit monoclonal antibody to β-TrCP (11984S; Cell Signaling Technology, Inc. [CST]), and mouse monoclonal antibodies to IκB (4814; CST), p-IκB (9246; CST), and CKIIα (sc-373894; SCB) were used at a 1:5,000 dilution.

    Techniques: Transfection, Expressing, Mutagenesis, Incubation, Generated, Plasmid Preparation

    Phosphorylation of NSP1 in rotavirus-infected cells. (A) Alignment of C-terminal sequences of NSP1 proteins, with the ILD boxed. (B) HT29 cells were infected with SA11-4F, SA11-5S, or OSU virus strains or monoreassortant SA11-L2 virus strains expressing OSU (SOF), KU (SKF), DS-1 (SDF), or RRV (SRF) NSP1. SA11-5S expresses a mutant form of NSP1 that lacks the 13 terminal residues of wild type SA11-4F NSP1. Lysates prepared from the infected cells at 15 h p.i. were analyzed by immunoblot assay using p-IκB antibody to detect p-NSP1 and PCNA antibody. To detect total NSP1, lysates from mock-, SA11-4F-, and SA11-5S-infected cells were probed with antibody against SA11-5S NSP1, lysates from OSU-, SOF-, SKF-, and SDF-infected cells were probed with antibody against OSU NSP1, and the lysate from RRV-infected cells was probed with antibody against RRV NSP1 ( 4 ).

    Journal: mBio

    Article Title: Rotavirus NSP1 Requires Casein Kinase II-Mediated Phosphorylation for Hijacking of Cullin-RING Ligases

    doi: 10.1128/mBio.01213-17

    Figure Lengend Snippet: Phosphorylation of NSP1 in rotavirus-infected cells. (A) Alignment of C-terminal sequences of NSP1 proteins, with the ILD boxed. (B) HT29 cells were infected with SA11-4F, SA11-5S, or OSU virus strains or monoreassortant SA11-L2 virus strains expressing OSU (SOF), KU (SKF), DS-1 (SDF), or RRV (SRF) NSP1. SA11-5S expresses a mutant form of NSP1 that lacks the 13 terminal residues of wild type SA11-4F NSP1. Lysates prepared from the infected cells at 15 h p.i. were analyzed by immunoblot assay using p-IκB antibody to detect p-NSP1 and PCNA antibody. To detect total NSP1, lysates from mock-, SA11-4F-, and SA11-5S-infected cells were probed with antibody against SA11-5S NSP1, lysates from OSU-, SOF-, SKF-, and SDF-infected cells were probed with antibody against OSU NSP1, and the lysate from RRV-infected cells was probed with antibody against RRV NSP1 ( 4 ).

    Article Snippet: Rabbit polyclonal antibodies to simian SA11-5S and porcine OSU NSP1 proteins ( ) and PCNA (sc-7907; Santa Cruz Biotech [SCB]), rabbit monoclonal antibody to β-TrCP (11984S; Cell Signaling Technology, Inc. [CST]), and mouse monoclonal antibodies to IκB (4814; CST), p-IκB (9246; CST), and CKIIα (sc-373894; SCB) were used at a 1:5,000 dilution.

    Techniques: Infection, Expressing, Mutagenesis

    Accumulation of p-NSP1, β-TrCP, and p-IκB during viral infection. HT29 cells were mock infected or infected with OSU rotavirus (MOI of 5) and harvested from 2 to 10 h p.i. Cellular lysates were resolved by gel electrophoresis and transferred onto nitrocellulose membranes. Blots were probed with antibodies specific for NSP1, p-NSP1, β-TrCP, p-IκB, and PCNA.

    Journal: mBio

    Article Title: Rotavirus NSP1 Requires Casein Kinase II-Mediated Phosphorylation for Hijacking of Cullin-RING Ligases

    doi: 10.1128/mBio.01213-17

    Figure Lengend Snippet: Accumulation of p-NSP1, β-TrCP, and p-IκB during viral infection. HT29 cells were mock infected or infected with OSU rotavirus (MOI of 5) and harvested from 2 to 10 h p.i. Cellular lysates were resolved by gel electrophoresis and transferred onto nitrocellulose membranes. Blots were probed with antibodies specific for NSP1, p-NSP1, β-TrCP, p-IκB, and PCNA.

    Article Snippet: Rabbit polyclonal antibodies to simian SA11-5S and porcine OSU NSP1 proteins ( ) and PCNA (sc-7907; Santa Cruz Biotech [SCB]), rabbit monoclonal antibody to β-TrCP (11984S; Cell Signaling Technology, Inc. [CST]), and mouse monoclonal antibodies to IκB (4814; CST), p-IκB (9246; CST), and CKIIα (sc-373894; SCB) were used at a 1:5,000 dilution.

    Techniques: Infection, Nucleic Acid Electrophoresis

    Effect of CKII inhibition on NSP1 phosphorylation. (A) HEK293T cells were cotransfected with a vector expressing HALO-tagged NSP1 (H-NSP1) and a 10 or 50 μM siRNA pool targeting the CKII RNA or representing a scrambled siRNA control pool. At 48 h p.t., cells were collected and protein samples were analyzed by immunoblot assay with antibodies specific for the HALO tag, p-NSP1, CKIIα, and PCNA. Band intensities from two independent experiments using 50 mM concentration of siRNA pools were determined with an Azure digital imager. (B) Levels of OSU NSP1 phosphorylation were calculated by dividing p-NSP1 intensity values by those for H-NSP1 and normalizing the results to 100% for the control siRNA sample. (C) CKII levels were calculated by dividing CKII intensity values by those for PCNA and normalizing the results to 100% for the control siRNA sample. (D) HT29 cells were infected with OSU at an MOI of 5 and treated with TBB or DMSO at 2 h p.i. At 4 and 9 h p.i., cells were collected and lysed, and proteins in the samples were resolved by electrophoresis and examined by immunoblot assay with antibodies recognizing OSU NSP1, p-NSP1, p-IκB, β-TrCP, or PCNA. (E) PK15 cells were infected with OSU at an MOI of 5 and mock treated or treated with TBB. Cells were harvest at 9 h p.i., and proteins in the samples were examined by immunoblot assay with antibodies to p-NSP1, rotavirus VP6, and PCNA.

    Journal: mBio

    Article Title: Rotavirus NSP1 Requires Casein Kinase II-Mediated Phosphorylation for Hijacking of Cullin-RING Ligases

    doi: 10.1128/mBio.01213-17

    Figure Lengend Snippet: Effect of CKII inhibition on NSP1 phosphorylation. (A) HEK293T cells were cotransfected with a vector expressing HALO-tagged NSP1 (H-NSP1) and a 10 or 50 μM siRNA pool targeting the CKII RNA or representing a scrambled siRNA control pool. At 48 h p.t., cells were collected and protein samples were analyzed by immunoblot assay with antibodies specific for the HALO tag, p-NSP1, CKIIα, and PCNA. Band intensities from two independent experiments using 50 mM concentration of siRNA pools were determined with an Azure digital imager. (B) Levels of OSU NSP1 phosphorylation were calculated by dividing p-NSP1 intensity values by those for H-NSP1 and normalizing the results to 100% for the control siRNA sample. (C) CKII levels were calculated by dividing CKII intensity values by those for PCNA and normalizing the results to 100% for the control siRNA sample. (D) HT29 cells were infected with OSU at an MOI of 5 and treated with TBB or DMSO at 2 h p.i. At 4 and 9 h p.i., cells were collected and lysed, and proteins in the samples were resolved by electrophoresis and examined by immunoblot assay with antibodies recognizing OSU NSP1, p-NSP1, p-IκB, β-TrCP, or PCNA. (E) PK15 cells were infected with OSU at an MOI of 5 and mock treated or treated with TBB. Cells were harvest at 9 h p.i., and proteins in the samples were examined by immunoblot assay with antibodies to p-NSP1, rotavirus VP6, and PCNA.

    Article Snippet: Rabbit polyclonal antibodies to simian SA11-5S and porcine OSU NSP1 proteins ( ) and PCNA (sc-7907; Santa Cruz Biotech [SCB]), rabbit monoclonal antibody to β-TrCP (11984S; Cell Signaling Technology, Inc. [CST]), and mouse monoclonal antibodies to IκB (4814; CST), p-IκB (9246; CST), and CKIIα (sc-373894; SCB) were used at a 1:5,000 dilution.

    Techniques: Inhibition, Plasmid Preparation, Expressing, Concentration Assay, Infection, Electrophoresis

    Polη promotion of PCNA ubiquitination depends on PIP3 and PIP2, but not on PIP1. (A) Schematic structure of human Polη. Parts of PIP and UBZ sequences are shown. Amino-acid residues indicated by asterisks were replaced with alanines in the mutants. (B) Western blot analysis of FLAG-Polη-expressing cells. XP-V cells were transfected with the indicated plasmids for expression of FLAG-Polη (wt) or the indicated pip mutants, incubated for 24 h, irradiated with UV (15 J/m 2 ) and further incubated for 3 h. Whole-cell lysates (WCL) or chromatin fractions were subjected to western blotting with anti-PCNA, anti-Polη and anti-Lamin B (loading control) antibodies.

    Journal: Nucleic Acids Research

    Article Title: Different types of interaction between PCNA and PIP boxes contribute to distinct cellular functions of Y-family DNA polymerases

    doi: 10.1093/nar/gkv712

    Figure Lengend Snippet: Polη promotion of PCNA ubiquitination depends on PIP3 and PIP2, but not on PIP1. (A) Schematic structure of human Polη. Parts of PIP and UBZ sequences are shown. Amino-acid residues indicated by asterisks were replaced with alanines in the mutants. (B) Western blot analysis of FLAG-Polη-expressing cells. XP-V cells were transfected with the indicated plasmids for expression of FLAG-Polη (wt) or the indicated pip mutants, incubated for 24 h, irradiated with UV (15 J/m 2 ) and further incubated for 3 h. Whole-cell lysates (WCL) or chromatin fractions were subjected to western blotting with anti-PCNA, anti-Polη and anti-Lamin B (loading control) antibodies.

    Article Snippet: Cellular fractions were analysed by western blotting with anti-PCNA (Santa Cruz Biotechnology, sc-7907 or sc-56), anti-Polη , anti-Lamin B (Santa Cruz Biotechnology, sc-6216), anti-FLAG (M2 SIGMA, F1804) or anti-GFP (MBL, M048–3) antibodies.

    Techniques: Western Blot, Expressing, Transfection, Incubation, Irradiation

    Promotion of mono-ubiquitination of PCNA in cells by Polκ but not Polι. (A, B) Western blot analysis of Polκ-expressing cells. XP-V (A) and normal cells (B) were transfected with a plasmid to express GFP-Polκ or GFP-Polη (as a control). (C, D) Western blot analysis of Polι-expressing cells. XP-V (C) and normal cells (D) were transfected with a plasmid to express FLAG-Polι or FLAG-Polη (as a control). The transfected cells were incubated for 24 h, irradiated with UV (15 J/m 2 ) and further incubated for 3 h. Whole-cell lysates (WCL) were subjected to western blotting with anti-PCNA, anti-Lamin B (loading control) and anti-GFP or anti-FLAG antibodies.

    Journal: Nucleic Acids Research

    Article Title: Different types of interaction between PCNA and PIP boxes contribute to distinct cellular functions of Y-family DNA polymerases

    doi: 10.1093/nar/gkv712

    Figure Lengend Snippet: Promotion of mono-ubiquitination of PCNA in cells by Polκ but not Polι. (A, B) Western blot analysis of Polκ-expressing cells. XP-V (A) and normal cells (B) were transfected with a plasmid to express GFP-Polκ or GFP-Polη (as a control). (C, D) Western blot analysis of Polι-expressing cells. XP-V (C) and normal cells (D) were transfected with a plasmid to express FLAG-Polι or FLAG-Polη (as a control). The transfected cells were incubated for 24 h, irradiated with UV (15 J/m 2 ) and further incubated for 3 h. Whole-cell lysates (WCL) were subjected to western blotting with anti-PCNA, anti-Lamin B (loading control) and anti-GFP or anti-FLAG antibodies.

    Article Snippet: Cellular fractions were analysed by western blotting with anti-PCNA (Santa Cruz Biotechnology, sc-7907 or sc-56), anti-Polη , anti-Lamin B (Santa Cruz Biotechnology, sc-6216), anti-FLAG (M2 SIGMA, F1804) or anti-GFP (MBL, M048–3) antibodies.

    Techniques: Western Blot, Expressing, Transfection, Plasmid Preparation, Incubation, Irradiation

    DNA polymerase assays of Polη in a reconstituted system in vitro . (A) DNA replication reactions using singly primed M13 mp18 ssDNA were reconstituted with the indicated factors. The reaction products were resolved in 10% polyacrylamide gels containing 7 M urea, and visualized using a PhosphorImager. (B) Analysis of pip mutants of Polη. Indicated mutants were subjected to replication assays shown in (A) in the presence or absence of PCNA.

    Journal: Nucleic Acids Research

    Article Title: Different types of interaction between PCNA and PIP boxes contribute to distinct cellular functions of Y-family DNA polymerases

    doi: 10.1093/nar/gkv712

    Figure Lengend Snippet: DNA polymerase assays of Polη in a reconstituted system in vitro . (A) DNA replication reactions using singly primed M13 mp18 ssDNA were reconstituted with the indicated factors. The reaction products were resolved in 10% polyacrylamide gels containing 7 M urea, and visualized using a PhosphorImager. (B) Analysis of pip mutants of Polη. Indicated mutants were subjected to replication assays shown in (A) in the presence or absence of PCNA.

    Article Snippet: Cellular fractions were analysed by western blotting with anti-PCNA (Santa Cruz Biotechnology, sc-7907 or sc-56), anti-Polη , anti-Lamin B (Santa Cruz Biotechnology, sc-6216), anti-FLAG (M2 SIGMA, F1804) or anti-GFP (MBL, M048–3) antibodies.

    Techniques: In Vitro

    In vitro reconstitution of Polη–dependent PCNA ubiquitination. (A) Mono-ubiquitination reactions of PCNA were reconstituted with the indicated factors. Reaction products were analysed by western blotting with an anti-PCNA antibody. KR indicates the PCNA K164R mutant. (B) Titration of Polη and its pip mutants. Indicated mutants were subjected to the ubiquitination assays as shown in (A). (C) Relative amounts of ubiquitinated PCNA were measured from gel images of more than three independent experiments, and the average values are plotted in the graph. Error bars show SD.

    Journal: Nucleic Acids Research

    Article Title: Different types of interaction between PCNA and PIP boxes contribute to distinct cellular functions of Y-family DNA polymerases

    doi: 10.1093/nar/gkv712

    Figure Lengend Snippet: In vitro reconstitution of Polη–dependent PCNA ubiquitination. (A) Mono-ubiquitination reactions of PCNA were reconstituted with the indicated factors. Reaction products were analysed by western blotting with an anti-PCNA antibody. KR indicates the PCNA K164R mutant. (B) Titration of Polη and its pip mutants. Indicated mutants were subjected to the ubiquitination assays as shown in (A). (C) Relative amounts of ubiquitinated PCNA were measured from gel images of more than three independent experiments, and the average values are plotted in the graph. Error bars show SD.

    Article Snippet: Cellular fractions were analysed by western blotting with anti-PCNA (Santa Cruz Biotechnology, sc-7907 or sc-56), anti-Polη , anti-Lamin B (Santa Cruz Biotechnology, sc-6216), anti-FLAG (M2 SIGMA, F1804) or anti-GFP (MBL, M048–3) antibodies.

    Techniques: In Vitro, Western Blot, Mutagenesis, Titration

    Cellular functions of the motifs of Polη. (A) Co-localization of Polη with PCNA. XP-V cells were transiently transfected with plasmids encoding wild-type FLAG-Polη or the indicated mutants. After UV irradiation, FLAG-Polη and PCNA were visualized by immunostaining with anti-Polη and anti-PCNA antibodies, respectively. Nuclei were stained by Hoechst 33342. Scale bars represent 5 μm. Control experiments confirming expressions of FLAG-Polη were shown in Supplementary Figure S7. (B) UV sensitivities of XP-V cells stably expressing FLAG-Polη. Cells were irradiated with the indicated dose of UVC, incubated with 1 mM caffeine for 4 days, and their viabilities were measured. Error bars show SD from three independent experiments. (C) A model for a regulatory network for foci formation and the TLS function of Polη/ι/κ. Interactions of Polη/κ with PCNA, together with RAD6-(RAD18) 2 , leads to their accumulation by promoting mono-ubiquitination of PCNA around stalled 3′-OH ends. Interactions of Polη/ι/κ with mUb-PCNA via PIPs and UBDs stimulate DNA synthesis at stalled 3′-OH ends. See text for details.

    Journal: Nucleic Acids Research

    Article Title: Different types of interaction between PCNA and PIP boxes contribute to distinct cellular functions of Y-family DNA polymerases

    doi: 10.1093/nar/gkv712

    Figure Lengend Snippet: Cellular functions of the motifs of Polη. (A) Co-localization of Polη with PCNA. XP-V cells were transiently transfected with plasmids encoding wild-type FLAG-Polη or the indicated mutants. After UV irradiation, FLAG-Polη and PCNA were visualized by immunostaining with anti-Polη and anti-PCNA antibodies, respectively. Nuclei were stained by Hoechst 33342. Scale bars represent 5 μm. Control experiments confirming expressions of FLAG-Polη were shown in Supplementary Figure S7. (B) UV sensitivities of XP-V cells stably expressing FLAG-Polη. Cells were irradiated with the indicated dose of UVC, incubated with 1 mM caffeine for 4 days, and their viabilities were measured. Error bars show SD from three independent experiments. (C) A model for a regulatory network for foci formation and the TLS function of Polη/ι/κ. Interactions of Polη/κ with PCNA, together with RAD6-(RAD18) 2 , leads to their accumulation by promoting mono-ubiquitination of PCNA around stalled 3′-OH ends. Interactions of Polη/ι/κ with mUb-PCNA via PIPs and UBDs stimulate DNA synthesis at stalled 3′-OH ends. See text for details.

    Article Snippet: Cellular fractions were analysed by western blotting with anti-PCNA (Santa Cruz Biotechnology, sc-7907 or sc-56), anti-Polη , anti-Lamin B (Santa Cruz Biotechnology, sc-6216), anti-FLAG (M2 SIGMA, F1804) or anti-GFP (MBL, M048–3) antibodies.

    Techniques: Transfection, Irradiation, Immunostaining, Staining, Stable Transfection, Expressing, Incubation, DNA Synthesis

    Interactions between Y-family DNA polymerases and mUb-PCNA in vitro . (A–J) Analysis of DNA synthesis by Polη (A–F), Polι (G–H) and His-Polκ (I–J), as shown in Figure 3 , in the absence or presence of PCNA (designated as PCNA or +) or mUb-PCNA (designated as uPCNA or u). Concentrations of polymerases increase in the order 0.25, 0.5, and 1 nM (A–F and I) or 1, 2, and 4 nM (G–H), or remain constant at 1 nM (J).

    Journal: Nucleic Acids Research

    Article Title: Different types of interaction between PCNA and PIP boxes contribute to distinct cellular functions of Y-family DNA polymerases

    doi: 10.1093/nar/gkv712

    Figure Lengend Snippet: Interactions between Y-family DNA polymerases and mUb-PCNA in vitro . (A–J) Analysis of DNA synthesis by Polη (A–F), Polι (G–H) and His-Polκ (I–J), as shown in Figure 3 , in the absence or presence of PCNA (designated as PCNA or +) or mUb-PCNA (designated as uPCNA or u). Concentrations of polymerases increase in the order 0.25, 0.5, and 1 nM (A–F and I) or 1, 2, and 4 nM (G–H), or remain constant at 1 nM (J).

    Article Snippet: Cellular fractions were analysed by western blotting with anti-PCNA (Santa Cruz Biotechnology, sc-7907 or sc-56), anti-Polη , anti-Lamin B (Santa Cruz Biotechnology, sc-6216), anti-FLAG (M2 SIGMA, F1804) or anti-GFP (MBL, M048–3) antibodies.

    Techniques: In Vitro, DNA Synthesis

    SUMO modification of PCNA limits MMS-induced DNA DSB formation and effects DNA damage sensitivity. ( A ) Representative images of HeLa cells stably expressing FLAG, FLAG-PCNA or FLAG-PCNA-SUMO1 either untreated or 2 and 5 h after MMS treatment subjected to comet assay under neutral conditions. ( B ) Percentage of comet tail DNA, a measure of DNA DSBs, at 0.5, 1, 2, 3 and 5 h after MMS treatment were calculated from three independent experiments using three independent stable cell lines; error bars show standard deviations. Significant difference was detected between the levels of double stand breaks of FLAG FLAG-PCNA-SUMO1, FLAG-PCNA and FLAG-PCNA-SUMO1 expressing cell lines * P

    Journal: Nucleic Acids Research

    Article Title: Role of SUMO modification of human PCNA at stalled replication fork

    doi: 10.1093/nar/gks256

    Figure Lengend Snippet: SUMO modification of PCNA limits MMS-induced DNA DSB formation and effects DNA damage sensitivity. ( A ) Representative images of HeLa cells stably expressing FLAG, FLAG-PCNA or FLAG-PCNA-SUMO1 either untreated or 2 and 5 h after MMS treatment subjected to comet assay under neutral conditions. ( B ) Percentage of comet tail DNA, a measure of DNA DSBs, at 0.5, 1, 2, 3 and 5 h after MMS treatment were calculated from three independent experiments using three independent stable cell lines; error bars show standard deviations. Significant difference was detected between the levels of double stand breaks of FLAG FLAG-PCNA-SUMO1, FLAG-PCNA and FLAG-PCNA-SUMO1 expressing cell lines * P

    Article Snippet: Next, we also evaluated the endogenous PCNA SUMOylation in cells stably expressing FLAG-SUMO1 by FLAG immunoprecipitation followed by probing with anti-PCNA antibody ( B).

    Techniques: Modification, Stable Transfection, Expressing, Single Cell Gel Electrophoresis

    In vivo SUMO modification of human PCNA. ( A ) HEK293T cells were co-transfected with HA-PCNA, His-UBC9 and either FLAG-SUMO1, or FLAG-SUMO2, or FLAG-SUMO3. In 48 h, post-transfection cells were UV-treated (30 J/m 2 ) or mock-irradiated and, after 3 h lysed and immunoprecipitated on FLAG-beads. FLAG-SUMO precipitates were immunoblotted with anti-HA antibody to detect PCNA and the SUMO-modified forms of PCNA. The lower panel shows the anti-HA western blot of the lysates. ( B ) Cell lysates and FLAG immunoprecipitates from control HeLa S3 cells and HeLa S3 cells stably expressing FLAG-SUMO1 were immunoblotted with anti-PCNA antibody to detect SUMOylated forms of endogenous PCNA. ( C ) HEK293T cells were co-transfected with FLAG-SUMO1, His-UBC9 and either HA-PCNA or K164R PCNA and 48-h post transfection cells were lysed and immunoprecipitated on FLAG-beads. FLAG-SUMO1 precipitates were immunoblotted with anti-HA antibody to detect the effect of the K164R mutation on the SUMOylation of PCNA. ( D ) Interaction of PCNA with UBC9 was tested by co-expressing FLAG-PCNA and HA-UBC9 in HEK293 cells followed by FLAG immunoprecipitation and then by western blot analysis with anti-HA and anti-FLAG antibodies for UBC9 and PCNA, respectively.

    Journal: Nucleic Acids Research

    Article Title: Role of SUMO modification of human PCNA at stalled replication fork

    doi: 10.1093/nar/gks256

    Figure Lengend Snippet: In vivo SUMO modification of human PCNA. ( A ) HEK293T cells were co-transfected with HA-PCNA, His-UBC9 and either FLAG-SUMO1, or FLAG-SUMO2, or FLAG-SUMO3. In 48 h, post-transfection cells were UV-treated (30 J/m 2 ) or mock-irradiated and, after 3 h lysed and immunoprecipitated on FLAG-beads. FLAG-SUMO precipitates were immunoblotted with anti-HA antibody to detect PCNA and the SUMO-modified forms of PCNA. The lower panel shows the anti-HA western blot of the lysates. ( B ) Cell lysates and FLAG immunoprecipitates from control HeLa S3 cells and HeLa S3 cells stably expressing FLAG-SUMO1 were immunoblotted with anti-PCNA antibody to detect SUMOylated forms of endogenous PCNA. ( C ) HEK293T cells were co-transfected with FLAG-SUMO1, His-UBC9 and either HA-PCNA or K164R PCNA and 48-h post transfection cells were lysed and immunoprecipitated on FLAG-beads. FLAG-SUMO1 precipitates were immunoblotted with anti-HA antibody to detect the effect of the K164R mutation on the SUMOylation of PCNA. ( D ) Interaction of PCNA with UBC9 was tested by co-expressing FLAG-PCNA and HA-UBC9 in HEK293 cells followed by FLAG immunoprecipitation and then by western blot analysis with anti-HA and anti-FLAG antibodies for UBC9 and PCNA, respectively.

    Article Snippet: Next, we also evaluated the endogenous PCNA SUMOylation in cells stably expressing FLAG-SUMO1 by FLAG immunoprecipitation followed by probing with anti-PCNA antibody ( B).

    Techniques: In Vivo, Modification, Transfection, Irradiation, Immunoprecipitation, Western Blot, Stable Transfection, Expressing, Mutagenesis

    Effect of SUMO modification of PCNA on homologous recombination. ( A ) I-SceI-induced recombinations and ( B ) spontaneous recombinations were measured as GFP positive cells after expressing control FLAG, FLAG-SUMO1, FLAG-PCNA or FLAG-PCNA-SUMO1. Error bars show standard deviation of the data obtained from three independent experiments.

    Journal: Nucleic Acids Research

    Article Title: Role of SUMO modification of human PCNA at stalled replication fork

    doi: 10.1093/nar/gks256

    Figure Lengend Snippet: Effect of SUMO modification of PCNA on homologous recombination. ( A ) I-SceI-induced recombinations and ( B ) spontaneous recombinations were measured as GFP positive cells after expressing control FLAG, FLAG-SUMO1, FLAG-PCNA or FLAG-PCNA-SUMO1. Error bars show standard deviation of the data obtained from three independent experiments.

    Article Snippet: Next, we also evaluated the endogenous PCNA SUMOylation in cells stably expressing FLAG-SUMO1 by FLAG immunoprecipitation followed by probing with anti-PCNA antibody ( B).

    Techniques: Modification, Homologous Recombination, Expressing, Standard Deviation

    In vitro SUMO modification of human PCNA. ( A ) in vitro SUMOylation reaction of human PCNA (40 nM) was carried out in the presence of purified SAE1/2 (10 nM), Ubc9 (100 nM), RFC (10 nM) nicked PUC19 plasmid DNA (2 nM) and either GST-SUMO1, or GST-SUMO2, or GST-SUMO3, or SUMO1, or SUMO2, or SUMO3 (500 nM) at 37°C for 60 min. Samples containing unmodified and SUMOylated PCNA were separated on 10% denaturing polyacrylamide gel and visualized by western blot using anti-PCNA antibody. Structure of human PCNA from the front ( B ) and side ( C ) views; surface lysine residues are represented by red spheres (K117, K138, K164, K168, K181, K190, K240, K248 and K254). PCNA structures showing the surface lysine residues were generated using the PyMOL version 0.96 by DeLano scientific ( http.//www.pymolsourceforge.net ). ( D ) Wild-type and lysine point-mutant PCNA samples were subjected to in vitro SUMOylation reaction as described above. ( E ) In vitro SUMOylation and ubiquitylation reactions of PCNA were compared in the absence or presence of combinations of RFC and nicked plasmid DNA as indicated.

    Journal: Nucleic Acids Research

    Article Title: Role of SUMO modification of human PCNA at stalled replication fork

    doi: 10.1093/nar/gks256

    Figure Lengend Snippet: In vitro SUMO modification of human PCNA. ( A ) in vitro SUMOylation reaction of human PCNA (40 nM) was carried out in the presence of purified SAE1/2 (10 nM), Ubc9 (100 nM), RFC (10 nM) nicked PUC19 plasmid DNA (2 nM) and either GST-SUMO1, or GST-SUMO2, or GST-SUMO3, or SUMO1, or SUMO2, or SUMO3 (500 nM) at 37°C for 60 min. Samples containing unmodified and SUMOylated PCNA were separated on 10% denaturing polyacrylamide gel and visualized by western blot using anti-PCNA antibody. Structure of human PCNA from the front ( B ) and side ( C ) views; surface lysine residues are represented by red spheres (K117, K138, K164, K168, K181, K190, K240, K248 and K254). PCNA structures showing the surface lysine residues were generated using the PyMOL version 0.96 by DeLano scientific ( http.//www.pymolsourceforge.net ). ( D ) Wild-type and lysine point-mutant PCNA samples were subjected to in vitro SUMOylation reaction as described above. ( E ) In vitro SUMOylation and ubiquitylation reactions of PCNA were compared in the absence or presence of combinations of RFC and nicked plasmid DNA as indicated.

    Article Snippet: Next, we also evaluated the endogenous PCNA SUMOylation in cells stably expressing FLAG-SUMO1 by FLAG immunoprecipitation followed by probing with anti-PCNA antibody ( B).

    Techniques: In Vitro, Modification, Purification, Plasmid Preparation, Western Blot, Generated, Mutagenesis

    Effect of PCNA-SUMO1 fusion protein on translesion synthesis polymerases and qualitative analysis of PCNA-SUMO1 fusion protein. ( A ) Schematic representation of the fusion of SUMO1 at the C-terminus of a FLAG-tagged PCNA and the control vectors expressing only FLAG, FLAG-SUMO1 or FLAG-PCNA. ( B ) DNA polymerase reactions were carried out using various TLS DNA polymerases on DNA substrate containing biotin–streptavidin at both ends generated by annealing a 75-nt long oligonucleotide template to a 5′ labelled 27-nt primer DNA in the presence of RFC and either PCNA or PCNA-SUMO1. The reaction products were analysed on 10% polyacrylamide gels containing 8 M urea, and the DNA bands were visualized by autoradiography. ( C ) HeLa cells stably expressing FLAG-PCNA and FLAG-PCNA-SUMO1 were pulse labelled with 10 µM BrdU for 1 h and immunostained with antibodies against FLAG (red) and BrdU (green). ( D ) Stable cell lines expressing FLAG-control, FLAG-PCNA and FLAG-PCNA-SUMO1 were subjected to flow cytometric analysis. Cell number is plotted on the y axis; DNA content on the x axis. Black, G1 peak; grey, G2/M peak; white, S phase fraction.

    Journal: Nucleic Acids Research

    Article Title: Role of SUMO modification of human PCNA at stalled replication fork

    doi: 10.1093/nar/gks256

    Figure Lengend Snippet: Effect of PCNA-SUMO1 fusion protein on translesion synthesis polymerases and qualitative analysis of PCNA-SUMO1 fusion protein. ( A ) Schematic representation of the fusion of SUMO1 at the C-terminus of a FLAG-tagged PCNA and the control vectors expressing only FLAG, FLAG-SUMO1 or FLAG-PCNA. ( B ) DNA polymerase reactions were carried out using various TLS DNA polymerases on DNA substrate containing biotin–streptavidin at both ends generated by annealing a 75-nt long oligonucleotide template to a 5′ labelled 27-nt primer DNA in the presence of RFC and either PCNA or PCNA-SUMO1. The reaction products were analysed on 10% polyacrylamide gels containing 8 M urea, and the DNA bands were visualized by autoradiography. ( C ) HeLa cells stably expressing FLAG-PCNA and FLAG-PCNA-SUMO1 were pulse labelled with 10 µM BrdU for 1 h and immunostained with antibodies against FLAG (red) and BrdU (green). ( D ) Stable cell lines expressing FLAG-control, FLAG-PCNA and FLAG-PCNA-SUMO1 were subjected to flow cytometric analysis. Cell number is plotted on the y axis; DNA content on the x axis. Black, G1 peak; grey, G2/M peak; white, S phase fraction.

    Article Snippet: Next, we also evaluated the endogenous PCNA SUMOylation in cells stably expressing FLAG-SUMO1 by FLAG immunoprecipitation followed by probing with anti-PCNA antibody ( B).

    Techniques: Translesion Synthesis, Expressing, Generated, Autoradiography, Stable Transfection, Flow Cytometry

    SUMO modification of PCNA reduces the accumulation of γH2AX foci. ( A ) HeLa cells stably expressing FLAG, FLAG-PCNA or FLAG-PCNA-SUMO1 were fixed 3 h after mock or ( B ) MMS treatments and stained with anti-FLAG (green), anti-γH2AX (red) and DAPI (blue). ( C ) Percentage of cell populations that showed more than two foci for γH2AX at 0.5, 1, 2, 3 and 5 h after MMS treatment was calculated from three independent experiments; error bars show standard deviations. ( D ) Effect of the expression of K164R PCNA in RAD18 −/− HCT116 cells was revealed by γH2AX staining 3 h after MMS treatment as described above.

    Journal: Nucleic Acids Research

    Article Title: Role of SUMO modification of human PCNA at stalled replication fork

    doi: 10.1093/nar/gks256

    Figure Lengend Snippet: SUMO modification of PCNA reduces the accumulation of γH2AX foci. ( A ) HeLa cells stably expressing FLAG, FLAG-PCNA or FLAG-PCNA-SUMO1 were fixed 3 h after mock or ( B ) MMS treatments and stained with anti-FLAG (green), anti-γH2AX (red) and DAPI (blue). ( C ) Percentage of cell populations that showed more than two foci for γH2AX at 0.5, 1, 2, 3 and 5 h after MMS treatment was calculated from three independent experiments; error bars show standard deviations. ( D ) Effect of the expression of K164R PCNA in RAD18 −/− HCT116 cells was revealed by γH2AX staining 3 h after MMS treatment as described above.

    Article Snippet: Next, we also evaluated the endogenous PCNA SUMOylation in cells stably expressing FLAG-SUMO1 by FLAG immunoprecipitation followed by probing with anti-PCNA antibody ( B).

    Techniques: Modification, Stable Transfection, Expressing, Staining

    Rad18 interacts with BPLF1 but does not compete with PCNA for a common binding site on BPLF1. (A) BPLF1 interacts with Rad18 in vitro . BPLF1 and Rad6/Rad18 protein complex expressed and purified from E. coli were incubated together at equimolar concentrations

    Journal: Journal of Virology

    Article Title: The Rad6/18 Ubiquitin Complex Interacts with the Epstein-Barr Virus Deubiquitinating Enzyme, BPLF1, and Contributes to Virus Infectivity

    doi: 10.1128/JVI.00536-14

    Figure Lengend Snippet: Rad18 interacts with BPLF1 but does not compete with PCNA for a common binding site on BPLF1. (A) BPLF1 interacts with Rad18 in vitro . BPLF1 and Rad6/Rad18 protein complex expressed and purified from E. coli were incubated together at equimolar concentrations

    Article Snippet: PCNA was detected with PCNA antibody (Santa Cruz Biotechnology), Rad18 was detected with Rad18 antibody (Bethyl Laboratories), Rad6 was detected with anti-Rad6 antibody (Bethyl Laboratories), and FLAG-tagged ubiquitin was detected with FLAG M2 antibody (Sigma).

    Techniques: Binding Assay, In Vitro, Purification, Incubation

    Rad18 interacts with BPLF1 in vivo . (A) H1299 cells were transfected with FLAG-tagged BPLF1 1-246. Lysates were immunoprecipitated (IP) with anti-FLAG antibody, followed by immunoblotting with FLAG, Rad18, and PCNA antibodies. Whole-cell lysates were

    Journal: Journal of Virology

    Article Title: The Rad6/18 Ubiquitin Complex Interacts with the Epstein-Barr Virus Deubiquitinating Enzyme, BPLF1, and Contributes to Virus Infectivity

    doi: 10.1128/JVI.00536-14

    Figure Lengend Snippet: Rad18 interacts with BPLF1 in vivo . (A) H1299 cells were transfected with FLAG-tagged BPLF1 1-246. Lysates were immunoprecipitated (IP) with anti-FLAG antibody, followed by immunoblotting with FLAG, Rad18, and PCNA antibodies. Whole-cell lysates were

    Article Snippet: PCNA was detected with PCNA antibody (Santa Cruz Biotechnology), Rad18 was detected with Rad18 antibody (Bethyl Laboratories), Rad6 was detected with anti-Rad6 antibody (Bethyl Laboratories), and FLAG-tagged ubiquitin was detected with FLAG M2 antibody (Sigma).

    Techniques: In Vivo, Transfection, Immunoprecipitation

    The telencephalon neurogenic zone contains three cell states. Telencephalic cross sections at the anteroposterior level indicated in the inset on the bottom left in h . a–d , Single optical section in a gfap:GFP transgenic brain section, immunostained for GFP (green), S100β (red), and PCNA (blue), depicting a close-up view on the ventricular surface at the midline between the two brain hemispheres. gfap: GFP and S100β are coexpressed in all radial glial cells (yellow in c ). Three cell states are visible: the majority of radial glial cells are noncycling (PCNA-negative; green arrow, state I cells). Some radial glial cells are PCNA positive (yellow arrow, state II cells). Some cells express only PCNA and no radial glial marker (red arrow, state III cells). e–g , State III cells express markers of neuroblasts. e , f , Expression of ascl1a revealed by in situ hybridization (black signal), revealing colocalization with PCNA (red). g , PSA-NCAM antibody staining (green) labels many state III cells, as shown by red arrows, revealing their identity of migrating neuroblasts. h , Overview of a gfap:GFP telencephalic section in a confocal stack projection, stained for GFP (green) and PCNA (blue). The arrows depict the telencephalic ventricle, located at the surface and at the midline. The boxed area depicts the region shown in a–g . The white line depicts the pallial–subpallial border region, in which mostly state III cells are located. The white dots depict the pallial ventricular region in which states I, II, and III are intermingled. This latter region is quantified in i and examined further in the next experiments. i , Relative proportions of state I, II, and III cells, counted in 2845 VZ cells of four brains. Scale bars: (in d ) a–d , 10 μm; (in g ) e–g , 10 μm; h , 100 μm.

    Journal: The Journal of Neuroscience

    Article Title: Notch Activity Levels Control the Balance between Quiescence and Recruitment of Adult Neural Stem Cells

    doi: 10.1523/JNEUROSCI.6170-09.2010

    Figure Lengend Snippet: The telencephalon neurogenic zone contains three cell states. Telencephalic cross sections at the anteroposterior level indicated in the inset on the bottom left in h . a–d , Single optical section in a gfap:GFP transgenic brain section, immunostained for GFP (green), S100β (red), and PCNA (blue), depicting a close-up view on the ventricular surface at the midline between the two brain hemispheres. gfap: GFP and S100β are coexpressed in all radial glial cells (yellow in c ). Three cell states are visible: the majority of radial glial cells are noncycling (PCNA-negative; green arrow, state I cells). Some radial glial cells are PCNA positive (yellow arrow, state II cells). Some cells express only PCNA and no radial glial marker (red arrow, state III cells). e–g , State III cells express markers of neuroblasts. e , f , Expression of ascl1a revealed by in situ hybridization (black signal), revealing colocalization with PCNA (red). g , PSA-NCAM antibody staining (green) labels many state III cells, as shown by red arrows, revealing their identity of migrating neuroblasts. h , Overview of a gfap:GFP telencephalic section in a confocal stack projection, stained for GFP (green) and PCNA (blue). The arrows depict the telencephalic ventricle, located at the surface and at the midline. The boxed area depicts the region shown in a–g . The white line depicts the pallial–subpallial border region, in which mostly state III cells are located. The white dots depict the pallial ventricular region in which states I, II, and III are intermingled. This latter region is quantified in i and examined further in the next experiments. i , Relative proportions of state I, II, and III cells, counted in 2845 VZ cells of four brains. Scale bars: (in d ) a–d , 10 μm; (in g ) e–g , 10 μm; h , 100 μm.

    Article Snippet: Primary antibodies directed against the following antigens were incubated 2 h at room temperature: BrdU (Roche), S100β (Dako), green fluorescent protein (GFP) (chicken; Aves Laboratories), proliferating cell nuclear antigen (PCNA) (Santa Cruz Biotechnology), MCM5 (kindly provided by Soojin Ryu), Hu (Invitrogen), polysialic acid-neural cell adhesion molecule (PSA-NCAM) (Millipore Bioscience Research Reagents), brain lipid binding protein (BLBP) (Millipore Bioscience Research Reagents), and Myc 9E10 clone (Sigma).

    Techniques: Transgenic Assay, Marker, Expressing, In Situ Hybridization, Staining

    In vivo transfection via lipofection and electroporation of dominant-negative constructs of the Notch pathway induces an increase of PCNA-positive cells. The ventricular zone of the telencephalon in adult fish was lipofected or electroporated with constructs in the pCS2 vector expressing eGFP ( a–c ) or was coelectroporated with pCS2–eGFP together with pCS2–dn-Su ( H ) ( d ), pCS2–deltastu ( e ), or pCS2–dn-maml1 ( f ). a and b depict the morphology and S100β expression ( b ) of transfected radial glia, which can be evaluated as individual cells. Transfected cells in c–f were assessed for their expression of PCNA (red) 2 d after electroporation; green arrows point to single GFP-positive cells, and yellow arrows point to GFP and PCNA double-positive cells. The percentage of GFP-positive cells also expressing PCNA is plotted in g for the different constructs. Note that all three dominant-negative proteins with Notch blockade activity increase cell cycle entry. In the case of dn-Su( H ) and Delta Stu , this increase reaches significance ( p = **0.0004 and *0.02, respectively, Fisher's exact test). n is the total number of cells counted of 5–10 brains per construct. Scale bars, 10 μm.

    Journal: The Journal of Neuroscience

    Article Title: Notch Activity Levels Control the Balance between Quiescence and Recruitment of Adult Neural Stem Cells

    doi: 10.1523/JNEUROSCI.6170-09.2010

    Figure Lengend Snippet: In vivo transfection via lipofection and electroporation of dominant-negative constructs of the Notch pathway induces an increase of PCNA-positive cells. The ventricular zone of the telencephalon in adult fish was lipofected or electroporated with constructs in the pCS2 vector expressing eGFP ( a–c ) or was coelectroporated with pCS2–eGFP together with pCS2–dn-Su ( H ) ( d ), pCS2–deltastu ( e ), or pCS2–dn-maml1 ( f ). a and b depict the morphology and S100β expression ( b ) of transfected radial glia, which can be evaluated as individual cells. Transfected cells in c–f were assessed for their expression of PCNA (red) 2 d after electroporation; green arrows point to single GFP-positive cells, and yellow arrows point to GFP and PCNA double-positive cells. The percentage of GFP-positive cells also expressing PCNA is plotted in g for the different constructs. Note that all three dominant-negative proteins with Notch blockade activity increase cell cycle entry. In the case of dn-Su( H ) and Delta Stu , this increase reaches significance ( p = **0.0004 and *0.02, respectively, Fisher's exact test). n is the total number of cells counted of 5–10 brains per construct. Scale bars, 10 μm.

    Article Snippet: Primary antibodies directed against the following antigens were incubated 2 h at room temperature: BrdU (Roche), S100β (Dako), green fluorescent protein (GFP) (chicken; Aves Laboratories), proliferating cell nuclear antigen (PCNA) (Santa Cruz Biotechnology), MCM5 (kindly provided by Soojin Ryu), Hu (Invitrogen), polysialic acid-neural cell adhesion molecule (PSA-NCAM) (Millipore Bioscience Research Reagents), brain lipid binding protein (BLBP) (Millipore Bioscience Research Reagents), and Myc 9E10 clone (Sigma).

    Techniques: In Vivo, Transfection, Electroporation, Dominant Negative Mutation, Construct, Fluorescence In Situ Hybridization, Plasmid Preparation, Expressing, Activity Assay

    Notch maintains quiescence in cells neighboring active progenitors. DAPT-treated ( b , d , i–k ) or vehicle-treated ( a , c , f–h ) fish were compared. e , Overview of a telencephalic cross section. Black rectangles depict regions enlarged in a–d . a , b , Medial ventricular area, containing dividing progenitors (PCNA, red) under normal conditions ( a ) and a higher density of PCNA-positive cells after DAPT ( b ). c , d , Dorsolateral area of the telencephalon (as boxed in e ; medial is to the top and lateral to the bottom) containing few PCNA-positive cells under normal conditions ( c ) and a slight increase of PCNA-positive cells after DAPT ( d ). The proportion of PCNA-positive cells reaches a higher level in medial regions under Notch-blocking conditions. f–k , Dorsomedial region harboring a density of PCNA-positive cells comparable with the region in a . BrdU was administered twice within 3 h, and 1 d later, the fish were treated with vehicle ( f–h ) or DAPT water ( i–k ) for 2 d. Cells neighboring BrdU-positive cells were analyzed for PCNA expression. Few BrdU-labeled cells in control brains are in contact with newly dividing, PCNA-positive cells (white arrow, f , g ), and many BrdU-positive cells (some of them are still PCNA positive; yellow) have no PCNA-positive (red) neighbor (green arrows, f , h ). Most BrdU-labeled cells after DAPT are surrounded by PCNA-positive cells (arrows in i–k ), suggesting that Notch prevents neighbors of an already dividing cell to enter cell cycle. The amount of BrdU-positive cells is unchanged, suggesting that the cell cycle speed has not been changed by the DAPT treatment. l , m , BrdU-labeled cells, counted in two brains for each condition, were categorized in three groups according to their neighbors: no dividing neighbor, one dividing neighbor, and more than one dividing neighbor. The proportion of cells belonging to each category is represented, and the error bars represent the SEM, in which n is number of brains. The blue bar of each category represents vehicle-treated and the yellow bar DAPT-treated animals. l , In DAPT-treated animals, the proportion of BrdU-positive, 3-d-labeled cells without dividing neighbor is decreased, whereas the proportion of BrdU-labeled cells with more than one neighbor is increased (one-way ANOVA for repeated measurements; 6 sections from 2 control brains and 4 sections from 2 DAPT brains; ** p

    Journal: The Journal of Neuroscience

    Article Title: Notch Activity Levels Control the Balance between Quiescence and Recruitment of Adult Neural Stem Cells

    doi: 10.1523/JNEUROSCI.6170-09.2010

    Figure Lengend Snippet: Notch maintains quiescence in cells neighboring active progenitors. DAPT-treated ( b , d , i–k ) or vehicle-treated ( a , c , f–h ) fish were compared. e , Overview of a telencephalic cross section. Black rectangles depict regions enlarged in a–d . a , b , Medial ventricular area, containing dividing progenitors (PCNA, red) under normal conditions ( a ) and a higher density of PCNA-positive cells after DAPT ( b ). c , d , Dorsolateral area of the telencephalon (as boxed in e ; medial is to the top and lateral to the bottom) containing few PCNA-positive cells under normal conditions ( c ) and a slight increase of PCNA-positive cells after DAPT ( d ). The proportion of PCNA-positive cells reaches a higher level in medial regions under Notch-blocking conditions. f–k , Dorsomedial region harboring a density of PCNA-positive cells comparable with the region in a . BrdU was administered twice within 3 h, and 1 d later, the fish were treated with vehicle ( f–h ) or DAPT water ( i–k ) for 2 d. Cells neighboring BrdU-positive cells were analyzed for PCNA expression. Few BrdU-labeled cells in control brains are in contact with newly dividing, PCNA-positive cells (white arrow, f , g ), and many BrdU-positive cells (some of them are still PCNA positive; yellow) have no PCNA-positive (red) neighbor (green arrows, f , h ). Most BrdU-labeled cells after DAPT are surrounded by PCNA-positive cells (arrows in i–k ), suggesting that Notch prevents neighbors of an already dividing cell to enter cell cycle. The amount of BrdU-positive cells is unchanged, suggesting that the cell cycle speed has not been changed by the DAPT treatment. l , m , BrdU-labeled cells, counted in two brains for each condition, were categorized in three groups according to their neighbors: no dividing neighbor, one dividing neighbor, and more than one dividing neighbor. The proportion of cells belonging to each category is represented, and the error bars represent the SEM, in which n is number of brains. The blue bar of each category represents vehicle-treated and the yellow bar DAPT-treated animals. l , In DAPT-treated animals, the proportion of BrdU-positive, 3-d-labeled cells without dividing neighbor is decreased, whereas the proportion of BrdU-labeled cells with more than one neighbor is increased (one-way ANOVA for repeated measurements; 6 sections from 2 control brains and 4 sections from 2 DAPT brains; ** p

    Article Snippet: Primary antibodies directed against the following antigens were incubated 2 h at room temperature: BrdU (Roche), S100β (Dako), green fluorescent protein (GFP) (chicken; Aves Laboratories), proliferating cell nuclear antigen (PCNA) (Santa Cruz Biotechnology), MCM5 (kindly provided by Soojin Ryu), Hu (Invitrogen), polysialic acid-neural cell adhesion molecule (PSA-NCAM) (Millipore Bioscience Research Reagents), brain lipid binding protein (BLBP) (Millipore Bioscience Research Reagents), and Myc 9E10 clone (Sigma).

    Techniques: Fluorescence In Situ Hybridization, Blocking Assay, Expressing, Labeling

    Expression of notch3 and the Notch reporter transgenic line highlight state I cells, whereas proliferating progenitors express deltaA . a–c , notch3a in situ hybridization (red signal) and MCM5 (green, proliferation marker) in the midline of the telencephalon showing a complementary expression: regions with dense expression of MCM5 express lower levels of notch3 (white arrow in a ), and high magnifications ( b , c ) reveal a complementary expression at the single-cell level. d , Cross sections, as a projection of a confocal stack, of a TP1bglob:gfp transgenic telencephalon, highlighting activation of RBPJ targets/canonical Notch signaling (green) along the VZ (the VZ is underlined by the white dotted line). e , GFP-positive cells colocalize with S100β (blue). Only a few (6%) weak GFP-positive cells (arrowhead) colocalize with S100β/PCNA double-positive cells (PCNA in red). f , g , Dividing cells marked by a BrdU pulse 6 h before analysis (orange, f ) or by PCNA (red, g ) express the Notch-ligand DeltaA (blue arrows), as visible by in situ hybridization ( f , blue signal) and in the deltaA:gfp transgenic line ( g , green; 95% of the PCNA-positive cells are GFP positive). The green arrow in g depicts also deltaA:GFP cells that are PCNA negative (64% of the GFP-positive cells are PCNA negative). h , Expression of deltaA:GFP (green) compared with S100β-positive radial glia (red). Green arrows point to single GFP-positive cells, and red arrows point to cells expressing S100β only. The majority of labeled cells are double-positive cells and appear yellow in the first panel (yellow arrows, 75% of deltaA:GFP cells are S100β positive). Scale bars: a , d , 100 μm; c , e , h , 10 μm; f , g , 50 μm.

    Journal: The Journal of Neuroscience

    Article Title: Notch Activity Levels Control the Balance between Quiescence and Recruitment of Adult Neural Stem Cells

    doi: 10.1523/JNEUROSCI.6170-09.2010

    Figure Lengend Snippet: Expression of notch3 and the Notch reporter transgenic line highlight state I cells, whereas proliferating progenitors express deltaA . a–c , notch3a in situ hybridization (red signal) and MCM5 (green, proliferation marker) in the midline of the telencephalon showing a complementary expression: regions with dense expression of MCM5 express lower levels of notch3 (white arrow in a ), and high magnifications ( b , c ) reveal a complementary expression at the single-cell level. d , Cross sections, as a projection of a confocal stack, of a TP1bglob:gfp transgenic telencephalon, highlighting activation of RBPJ targets/canonical Notch signaling (green) along the VZ (the VZ is underlined by the white dotted line). e , GFP-positive cells colocalize with S100β (blue). Only a few (6%) weak GFP-positive cells (arrowhead) colocalize with S100β/PCNA double-positive cells (PCNA in red). f , g , Dividing cells marked by a BrdU pulse 6 h before analysis (orange, f ) or by PCNA (red, g ) express the Notch-ligand DeltaA (blue arrows), as visible by in situ hybridization ( f , blue signal) and in the deltaA:gfp transgenic line ( g , green; 95% of the PCNA-positive cells are GFP positive). The green arrow in g depicts also deltaA:GFP cells that are PCNA negative (64% of the GFP-positive cells are PCNA negative). h , Expression of deltaA:GFP (green) compared with S100β-positive radial glia (red). Green arrows point to single GFP-positive cells, and red arrows point to cells expressing S100β only. The majority of labeled cells are double-positive cells and appear yellow in the first panel (yellow arrows, 75% of deltaA:GFP cells are S100β positive). Scale bars: a , d , 100 μm; c , e , h , 10 μm; f , g , 50 μm.

    Article Snippet: Primary antibodies directed against the following antigens were incubated 2 h at room temperature: BrdU (Roche), S100β (Dako), green fluorescent protein (GFP) (chicken; Aves Laboratories), proliferating cell nuclear antigen (PCNA) (Santa Cruz Biotechnology), MCM5 (kindly provided by Soojin Ryu), Hu (Invitrogen), polysialic acid-neural cell adhesion molecule (PSA-NCAM) (Millipore Bioscience Research Reagents), brain lipid binding protein (BLBP) (Millipore Bioscience Research Reagents), and Myc 9E10 clone (Sigma).

    Techniques: Expressing, Transgenic Assay, In Situ Hybridization, Marker, Activation Assay, Labeling

    State II cells endogenously transit to state I and back to state II, according to Notch activity. a–e , Cross section of a gfap:GFP transgenic fish (GFP in blue) treated with BrdU for 5 d and killed 40 d after the last injection. Some BrdU-labeled cells (green) remain in the ventricular area but are PCNA (red) negative, as pointed to by the arrow, indicating that they have entered state I after having divided. f–j , TP1bglob:gfp transgenic fish treated with BrdU for 5 d and killed 2.5 months later. f , Projection of several confocal planes, overview of the telencephalic midline: Many BrdU+ cells (blue) have exited from the ventricular zone containing PCNA+ cells (red) and have entered the parenchyma (blue arrow). g–j , Single optical section showing the area boxed in f . Some BrdU+, PCNA-negative cells remaining in the ventricular area are GFP+ (white arrow), indicating that they have entered state I and are under high Notch signaling. k–o , WT fish were injected with BrdU for 5 d and treated 3 weeks later for 2 d with DAPT or control water. Brains were stained for BrdU (green) and PCNA (red). o , The proportion of BrdU+, PCNA+ cells within the ventricular BrdU+ population was calculated in two control and two DAPT-treated brains. After DAPT treatment, this proportion is increased by 5 times compared to the control, indicating that Notch signaling had kept label-retaining cells quiescent. Error bars represent SEM, n = 2 with a total number of 217 control and 235 DAPT-treated BrdU+ cells, p

    Journal: The Journal of Neuroscience

    Article Title: Notch Activity Levels Control the Balance between Quiescence and Recruitment of Adult Neural Stem Cells

    doi: 10.1523/JNEUROSCI.6170-09.2010

    Figure Lengend Snippet: State II cells endogenously transit to state I and back to state II, according to Notch activity. a–e , Cross section of a gfap:GFP transgenic fish (GFP in blue) treated with BrdU for 5 d and killed 40 d after the last injection. Some BrdU-labeled cells (green) remain in the ventricular area but are PCNA (red) negative, as pointed to by the arrow, indicating that they have entered state I after having divided. f–j , TP1bglob:gfp transgenic fish treated with BrdU for 5 d and killed 2.5 months later. f , Projection of several confocal planes, overview of the telencephalic midline: Many BrdU+ cells (blue) have exited from the ventricular zone containing PCNA+ cells (red) and have entered the parenchyma (blue arrow). g–j , Single optical section showing the area boxed in f . Some BrdU+, PCNA-negative cells remaining in the ventricular area are GFP+ (white arrow), indicating that they have entered state I and are under high Notch signaling. k–o , WT fish were injected with BrdU for 5 d and treated 3 weeks later for 2 d with DAPT or control water. Brains were stained for BrdU (green) and PCNA (red). o , The proportion of BrdU+, PCNA+ cells within the ventricular BrdU+ population was calculated in two control and two DAPT-treated brains. After DAPT treatment, this proportion is increased by 5 times compared to the control, indicating that Notch signaling had kept label-retaining cells quiescent. Error bars represent SEM, n = 2 with a total number of 217 control and 235 DAPT-treated BrdU+ cells, p

    Article Snippet: Primary antibodies directed against the following antigens were incubated 2 h at room temperature: BrdU (Roche), S100β (Dako), green fluorescent protein (GFP) (chicken; Aves Laboratories), proliferating cell nuclear antigen (PCNA) (Santa Cruz Biotechnology), MCM5 (kindly provided by Soojin Ryu), Hu (Invitrogen), polysialic acid-neural cell adhesion molecule (PSA-NCAM) (Millipore Bioscience Research Reagents), brain lipid binding protein (BLBP) (Millipore Bioscience Research Reagents), and Myc 9E10 clone (Sigma).

    Techniques: Activity Assay, Transgenic Assay, Fluorescence In Situ Hybridization, Injection, Labeling, Staining

    Notch blocking induces an increased number of neuroblasts. Cross sections through the telencephalon of wild-type animals, treated with vehicle only ( a–e ) or with DAPT ( f–j ). a , b , f , g , In situ hybridization with the ascl1a antisense probe (blue in a and f and black in b and g ). DAPT treatment induces an increased number of dividing cells undergoing neurogenesis. c–e , h–j , PSA-NCAM (green), PCNA (red), and S100β (blue), merged in e and j . DAPT treatment induces an increased number of PCNA+, PSA-NCAM+ cells (state III cells, white arrow) and of cells only positive for PSA-NCAM (likely differentiating neurons, green arrow). S100β, PCNA, PSA-NCAM triple-labeled cells are also increased under DAPT conditions (arrowhead), suggesting an accelerated switch from state II to state III. k , Dividing cells in 2 control- and 2 DAPT-treated fish were traced for 4 d after BrdU injection. The proportion of BrdU+, Hu+ cells is similar in control and treated brains, indicating that the reactivation of state I cells by DAPT is followed by normal neuronal maturation, and that consequently the net number of generated neurons is increased. Scale bars: g , 50 μm; j , 10 μm.

    Journal: The Journal of Neuroscience

    Article Title: Notch Activity Levels Control the Balance between Quiescence and Recruitment of Adult Neural Stem Cells

    doi: 10.1523/JNEUROSCI.6170-09.2010

    Figure Lengend Snippet: Notch blocking induces an increased number of neuroblasts. Cross sections through the telencephalon of wild-type animals, treated with vehicle only ( a–e ) or with DAPT ( f–j ). a , b , f , g , In situ hybridization with the ascl1a antisense probe (blue in a and f and black in b and g ). DAPT treatment induces an increased number of dividing cells undergoing neurogenesis. c–e , h–j , PSA-NCAM (green), PCNA (red), and S100β (blue), merged in e and j . DAPT treatment induces an increased number of PCNA+, PSA-NCAM+ cells (state III cells, white arrow) and of cells only positive for PSA-NCAM (likely differentiating neurons, green arrow). S100β, PCNA, PSA-NCAM triple-labeled cells are also increased under DAPT conditions (arrowhead), suggesting an accelerated switch from state II to state III. k , Dividing cells in 2 control- and 2 DAPT-treated fish were traced for 4 d after BrdU injection. The proportion of BrdU+, Hu+ cells is similar in control and treated brains, indicating that the reactivation of state I cells by DAPT is followed by normal neuronal maturation, and that consequently the net number of generated neurons is increased. Scale bars: g , 50 μm; j , 10 μm.

    Article Snippet: Primary antibodies directed against the following antigens were incubated 2 h at room temperature: BrdU (Roche), S100β (Dako), green fluorescent protein (GFP) (chicken; Aves Laboratories), proliferating cell nuclear antigen (PCNA) (Santa Cruz Biotechnology), MCM5 (kindly provided by Soojin Ryu), Hu (Invitrogen), polysialic acid-neural cell adhesion molecule (PSA-NCAM) (Millipore Bioscience Research Reagents), brain lipid binding protein (BLBP) (Millipore Bioscience Research Reagents), and Myc 9E10 clone (Sigma).

    Techniques: Blocking Assay, In Situ Hybridization, Labeling, Fluorescence In Situ Hybridization, Injection, Generated

    Immunohistochemical expression of p53 (A) and PCNA (B) in calcifying cystic odontogenic tumor (400X).

    Journal: International Journal of Molecular and Cellular Medicine

    Article Title: p53 and PCNA Expression in Keratocystic Odontogenic Tumors Compared with Selected Odontogenic Cysts

    doi:

    Figure Lengend Snippet: Immunohistochemical expression of p53 (A) and PCNA (B) in calcifying cystic odontogenic tumor (400X).

    Article Snippet: The slides were covered by monoclonal antibody for p53 (Clone D07, Isotype; IgG2b Kappa, DakoCytomation, Glostrup, Denmark) and PCNA (Clone PC10, Isotype; IgG2a Kappa, DakoCytomation, Glostrup, Denmark) for 1 h, then rinsed in tap water and placed in TBS for 5 min. After immunostaining, the slides were coun-terstained with Meyer's hematoxylin, mounted with entellan and coverslide and examined by light microscopy (Olympus BX41, Shibuya-Ku, Tokyo, Japan) under 400X magnification.

    Techniques: Immunohistochemistry, Expressing

    Immunohistochemical expression of p53 (A) and PCNA (B) in dentigerous cyst (400X) (400X).

    Journal: International Journal of Molecular and Cellular Medicine

    Article Title: p53 and PCNA Expression in Keratocystic Odontogenic Tumors Compared with Selected Odontogenic Cysts

    doi:

    Figure Lengend Snippet: Immunohistochemical expression of p53 (A) and PCNA (B) in dentigerous cyst (400X) (400X).

    Article Snippet: The slides were covered by monoclonal antibody for p53 (Clone D07, Isotype; IgG2b Kappa, DakoCytomation, Glostrup, Denmark) and PCNA (Clone PC10, Isotype; IgG2a Kappa, DakoCytomation, Glostrup, Denmark) for 1 h, then rinsed in tap water and placed in TBS for 5 min. After immunostaining, the slides were coun-terstained with Meyer's hematoxylin, mounted with entellan and coverslide and examined by light microscopy (Olympus BX41, Shibuya-Ku, Tokyo, Japan) under 400X magnification.

    Techniques: Immunohistochemistry, Expressing

    Immunohistochemical expression of p53 (A) and PCNA (B) in radicular cyst (400X).

    Journal: International Journal of Molecular and Cellular Medicine

    Article Title: p53 and PCNA Expression in Keratocystic Odontogenic Tumors Compared with Selected Odontogenic Cysts

    doi:

    Figure Lengend Snippet: Immunohistochemical expression of p53 (A) and PCNA (B) in radicular cyst (400X).

    Article Snippet: The slides were covered by monoclonal antibody for p53 (Clone D07, Isotype; IgG2b Kappa, DakoCytomation, Glostrup, Denmark) and PCNA (Clone PC10, Isotype; IgG2a Kappa, DakoCytomation, Glostrup, Denmark) for 1 h, then rinsed in tap water and placed in TBS for 5 min. After immunostaining, the slides were coun-terstained with Meyer's hematoxylin, mounted with entellan and coverslide and examined by light microscopy (Olympus BX41, Shibuya-Ku, Tokyo, Japan) under 400X magnification.

    Techniques: Immunohistochemistry, Expressing

    p53 and PCNA expression in basal and suprabasal layers in different odontogenic cysts

    Journal: International Journal of Molecular and Cellular Medicine

    Article Title: p53 and PCNA Expression in Keratocystic Odontogenic Tumors Compared with Selected Odontogenic Cysts

    doi:

    Figure Lengend Snippet: p53 and PCNA expression in basal and suprabasal layers in different odontogenic cysts

    Article Snippet: The slides were covered by monoclonal antibody for p53 (Clone D07, Isotype; IgG2b Kappa, DakoCytomation, Glostrup, Denmark) and PCNA (Clone PC10, Isotype; IgG2a Kappa, DakoCytomation, Glostrup, Denmark) for 1 h, then rinsed in tap water and placed in TBS for 5 min. After immunostaining, the slides were coun-terstained with Meyer's hematoxylin, mounted with entellan and coverslide and examined by light microscopy (Olympus BX41, Shibuya-Ku, Tokyo, Japan) under 400X magnification.

    Techniques: Expressing

    Immunohistochemical expression of p53 (A) and PCNA (B) in keratocystic odontogenic tumor (400X).

    Journal: International Journal of Molecular and Cellular Medicine

    Article Title: p53 and PCNA Expression in Keratocystic Odontogenic Tumors Compared with Selected Odontogenic Cysts

    doi:

    Figure Lengend Snippet: Immunohistochemical expression of p53 (A) and PCNA (B) in keratocystic odontogenic tumor (400X).

    Article Snippet: The slides were covered by monoclonal antibody for p53 (Clone D07, Isotype; IgG2b Kappa, DakoCytomation, Glostrup, Denmark) and PCNA (Clone PC10, Isotype; IgG2a Kappa, DakoCytomation, Glostrup, Denmark) for 1 h, then rinsed in tap water and placed in TBS for 5 min. After immunostaining, the slides were coun-terstained with Meyer's hematoxylin, mounted with entellan and coverslide and examined by light microscopy (Olympus BX41, Shibuya-Ku, Tokyo, Japan) under 400X magnification.

    Techniques: Immunohistochemistry, Expressing

    Ligase and Fen1 have a shorter residence time at RF than PCNA ( A ) Half of the nucleus (BA) was bleached by one single, 12 s bleach pulse in live cells expressing GFP-Ligase or GFP-Fen1 and RFP-PCNA, resulting in an almost complete depletion of Ligase and Fen1 from the unbleached nucleoplasm and RF, in contrast to PCNA. ( B ) Different residence times of PCNA, Ligase and Fen1 at RF shown by loss of fluorescence from unbleached RF immediately after bleaching (16–20 RF from five cells were analyzed in each case). ( C ) Fast equilibration of the remaining Ligase and Fen1 between bleached and unbleached half of the nucleus within a few seconds in contrast to a slow equilibration of PCNA over minutes (three cells each). Scale bar, 5 μm.

    Journal: Nucleic Acids Research

    Article Title: PCNA acts as a stationary loading platform for transiently interacting Okazaki fragment maturation proteins

    doi: 10.1093/nar/gki665

    Figure Lengend Snippet: Ligase and Fen1 have a shorter residence time at RF than PCNA ( A ) Half of the nucleus (BA) was bleached by one single, 12 s bleach pulse in live cells expressing GFP-Ligase or GFP-Fen1 and RFP-PCNA, resulting in an almost complete depletion of Ligase and Fen1 from the unbleached nucleoplasm and RF, in contrast to PCNA. ( B ) Different residence times of PCNA, Ligase and Fen1 at RF shown by loss of fluorescence from unbleached RF immediately after bleaching (16–20 RF from five cells were analyzed in each case). ( C ) Fast equilibration of the remaining Ligase and Fen1 between bleached and unbleached half of the nucleus within a few seconds in contrast to a slow equilibration of PCNA over minutes (three cells each). Scale bar, 5 μm.

    Article Snippet: Amounts of endogenous PCNA and Ligase as well as the respective GFP fusion were detected with anti-Ligase ( ) or anti-PCNA (clone PC10, Dako) antibodies.

    Techniques: Expressing, Fluorescence

    Fast reassociation of Ligase and Fen1 at active RF contrasts to slow assembly of PCNA at newly established RF ( A ) In S-phase cells expressing GFP-Ligase or GFP-Fen1 and RFP-PCNA, one RF was bleached (BA). Ligase and Fen1 showed recovery at the bleached RF within a few seconds, while reappearance of PCNA fluorescence occurred several minutes later (for quantification, see diagrams). ( B ) Detailed spatial (insets, top panel) and time overlay analyses (insets, lower panel) showing the fast recovery of Ligase (top panel, mid) at previously bleached RF (lower panel, middle). However, the shape of the recovered Ligase focus changes over time (lower panel, right). In contrast, delayed recovery of PCNA occurs only at a small portion of the Ligase-labeled RF (top panel, middle and right) and is mostly adjacent to the bleached RF (lower panel, left). ( C ) Schematic interpretation of the data with fast exchanging Ligase/Fen1 continuously labeling active and newly assembled replisomes, in contrast with PCNA reassembling mostly at newly established replisomes. ( D ) Model of PCNA (red) as a stationary loading platform for the sequential loading of replication enzymes (here, represented by Fen1 in green and Ligase in blue) at the lagging strand synthesis. For simplicity, only one replicating lagging strand and one Okazaki fragment are shown. Scale bar, 5 μm.

    Journal: Nucleic Acids Research

    Article Title: PCNA acts as a stationary loading platform for transiently interacting Okazaki fragment maturation proteins

    doi: 10.1093/nar/gki665

    Figure Lengend Snippet: Fast reassociation of Ligase and Fen1 at active RF contrasts to slow assembly of PCNA at newly established RF ( A ) In S-phase cells expressing GFP-Ligase or GFP-Fen1 and RFP-PCNA, one RF was bleached (BA). Ligase and Fen1 showed recovery at the bleached RF within a few seconds, while reappearance of PCNA fluorescence occurred several minutes later (for quantification, see diagrams). ( B ) Detailed spatial (insets, top panel) and time overlay analyses (insets, lower panel) showing the fast recovery of Ligase (top panel, mid) at previously bleached RF (lower panel, middle). However, the shape of the recovered Ligase focus changes over time (lower panel, right). In contrast, delayed recovery of PCNA occurs only at a small portion of the Ligase-labeled RF (top panel, middle and right) and is mostly adjacent to the bleached RF (lower panel, left). ( C ) Schematic interpretation of the data with fast exchanging Ligase/Fen1 continuously labeling active and newly assembled replisomes, in contrast with PCNA reassembling mostly at newly established replisomes. ( D ) Model of PCNA (red) as a stationary loading platform for the sequential loading of replication enzymes (here, represented by Fen1 in green and Ligase in blue) at the lagging strand synthesis. For simplicity, only one replicating lagging strand and one Okazaki fragment are shown. Scale bar, 5 μm.

    Article Snippet: Amounts of endogenous PCNA and Ligase as well as the respective GFP fusion were detected with anti-Ligase ( ) or anti-PCNA (clone PC10, Dako) antibodies.

    Techniques: Expressing, Fluorescence, Labeling

    Ligase has a higher nucleoplasmic fraction than PCNA ( A ) Confocal images of live C2C12 cells in S-phase transiently or stably expressing GFP-Ligase or GFP-PCNA. ( B ) Quantification of NP and RF-bound protein (green/red in the false color image) by determining their mean FI ( n = 5 cells with 5–7 z slices). Scale bar, 5 μm.

    Journal: Nucleic Acids Research

    Article Title: PCNA acts as a stationary loading platform for transiently interacting Okazaki fragment maturation proteins

    doi: 10.1093/nar/gki665

    Figure Lengend Snippet: Ligase has a higher nucleoplasmic fraction than PCNA ( A ) Confocal images of live C2C12 cells in S-phase transiently or stably expressing GFP-Ligase or GFP-PCNA. ( B ) Quantification of NP and RF-bound protein (green/red in the false color image) by determining their mean FI ( n = 5 cells with 5–7 z slices). Scale bar, 5 μm.

    Article Snippet: Amounts of endogenous PCNA and Ligase as well as the respective GFP fusion were detected with anti-Ligase ( ) or anti-PCNA (clone PC10, Dako) antibodies.

    Techniques: Stable Transfection, Expressing

    Ligase is less strongly associated to replication sites than PCNA ( A ) Time-lapse in situ salt extraction of non-S- and S-phase cells expressing GFP-Ligase and RFP-PCNA showing that GFP-Ligase is largely extracted after short permeabilization with Triton X-100, while RFP–PCNA remains associated at RF for many minutes (for quantification, see diagram). ( B ) Identical results were obtained for endogenous Ligase and PCNA, which were detected by immunofluorescence. Scale bar, 10 μm. ( C ) C2C12 cells were cotransfected with both GFP-PCNA and GFP-Ligase to ensure the same number of transfected cells and the same percentage of S-phase cells. Cells were permeabilized and extracted, and equal amounts of the cell extract and the insoluble cell pellet were analyzed by SDS–PAGE followed by western blotting. Whereas most of the endogenous Ligase and the respective GFP fusion protein were found in the soluble fraction, under the same conditions, endogenous PCNA and GFP-PCNA were only partially extracted from the pellet and found equally distributed in both fractions. In this direct comparison, PCNA showed a stronger, label-independent association with nuclear structures than Ligase.

    Journal: Nucleic Acids Research

    Article Title: PCNA acts as a stationary loading platform for transiently interacting Okazaki fragment maturation proteins

    doi: 10.1093/nar/gki665

    Figure Lengend Snippet: Ligase is less strongly associated to replication sites than PCNA ( A ) Time-lapse in situ salt extraction of non-S- and S-phase cells expressing GFP-Ligase and RFP-PCNA showing that GFP-Ligase is largely extracted after short permeabilization with Triton X-100, while RFP–PCNA remains associated at RF for many minutes (for quantification, see diagram). ( B ) Identical results were obtained for endogenous Ligase and PCNA, which were detected by immunofluorescence. Scale bar, 10 μm. ( C ) C2C12 cells were cotransfected with both GFP-PCNA and GFP-Ligase to ensure the same number of transfected cells and the same percentage of S-phase cells. Cells were permeabilized and extracted, and equal amounts of the cell extract and the insoluble cell pellet were analyzed by SDS–PAGE followed by western blotting. Whereas most of the endogenous Ligase and the respective GFP fusion protein were found in the soluble fraction, under the same conditions, endogenous PCNA and GFP-PCNA were only partially extracted from the pellet and found equally distributed in both fractions. In this direct comparison, PCNA showed a stronger, label-independent association with nuclear structures than Ligase.

    Article Snippet: Amounts of endogenous PCNA and Ligase as well as the respective GFP fusion were detected with anti-Ligase ( ) or anti-PCNA (clone PC10, Dako) antibodies.

    Techniques: In Situ, Expressing, Immunofluorescence, Transfection, SDS Page, Western Blot

    MutLα interacts with PCNA in solution. ( A and B ) Examples of equilibrium gel filtration profiles after injection of 10 μL 10 μM wild-type ( A ) or PMS2-Q721A MutLα ( B ) onto a Superdex 200 column equilibrated with 10 μM

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Interaction of proliferating cell nuclear antigen with PMS2 is required for MutLα activation and function in mismatch repair

    doi: 10.1073/pnas.1702561114

    Figure Lengend Snippet: MutLα interacts with PCNA in solution. ( A and B ) Examples of equilibrium gel filtration profiles after injection of 10 μL 10 μM wild-type ( A ) or PMS2-Q721A MutLα ( B ) onto a Superdex 200 column equilibrated with 10 μM

    Article Snippet: After blocking with 5% milk for 1 h at room temperature, the membrane was incubated overnight at 4 °C with primary antibody [rabbit anti-PMS2 (sc618), mouse anti-PCNA (sc56), or rabbit anti-MLH1 (sc582); Santa Cruz Biotechnology].

    Techniques: Filtration, Injection

    BS 3 cross-links MutLα MLH1-AAA and PCNA. Wild-type MutLα, MutLα MLH1-AAA, and MutLα PMS2-AAA were cross-linked with BS 3 in the absence or presence of PCNA as described in the legend to . The asterisk indicates the

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Interaction of proliferating cell nuclear antigen with PMS2 is required for MutLα activation and function in mismatch repair

    doi: 10.1073/pnas.1702561114

    Figure Lengend Snippet: BS 3 cross-links MutLα MLH1-AAA and PCNA. Wild-type MutLα, MutLα MLH1-AAA, and MutLα PMS2-AAA were cross-linked with BS 3 in the absence or presence of PCNA as described in the legend to . The asterisk indicates the

    Article Snippet: After blocking with 5% milk for 1 h at room temperature, the membrane was incubated overnight at 4 °C with primary antibody [rabbit anti-PMS2 (sc618), mouse anti-PCNA (sc56), or rabbit anti-MLH1 (sc582); Santa Cruz Biotechnology].

    Techniques:

    BS 3 cross-linking of MutLα and PCNA. ( A ) Wild-type, PMS2-Q721A, or PMS2-AAA MutLα was cross-linked with BS 3 ) and reaction products were resolved by SDS/PAGE and visualized with

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Interaction of proliferating cell nuclear antigen with PMS2 is required for MutLα activation and function in mismatch repair

    doi: 10.1073/pnas.1702561114

    Figure Lengend Snippet: BS 3 cross-linking of MutLα and PCNA. ( A ) Wild-type, PMS2-Q721A, or PMS2-AAA MutLα was cross-linked with BS 3 ) and reaction products were resolved by SDS/PAGE and visualized with

    Article Snippet: After blocking with 5% milk for 1 h at room temperature, the membrane was incubated overnight at 4 °C with primary antibody [rabbit anti-PMS2 (sc618), mouse anti-PCNA (sc56), or rabbit anti-MLH1 (sc582); Santa Cruz Biotechnology].

    Techniques: SDS Page

    PCNA- and DNA-dependent stimulation of MutLα ATPase is attenuated by amino acid substitutions within the PMS2 721 QRLIAP motif. ( A ) by 0.68 μM wild-type MutLα (blue circles), MutLα

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Interaction of proliferating cell nuclear antigen with PMS2 is required for MutLα activation and function in mismatch repair

    doi: 10.1073/pnas.1702561114

    Figure Lengend Snippet: PCNA- and DNA-dependent stimulation of MutLα ATPase is attenuated by amino acid substitutions within the PMS2 721 QRLIAP motif. ( A ) by 0.68 μM wild-type MutLα (blue circles), MutLα

    Article Snippet: After blocking with 5% milk for 1 h at room temperature, the membrane was incubated overnight at 4 °C with primary antibody [rabbit anti-PMS2 (sc618), mouse anti-PCNA (sc56), or rabbit anti-MLH1 (sc582); Santa Cruz Biotechnology].

    Techniques:

    PCNA stimulation of MutLα endonuclease activity on linear DNA depends on integrity of the PMS2 721 QRLIAP motif. 50 ng (280 fmol) wild type MutLα, PMS2-Q721A, or PMS2-AAA were incubated with a fluorescently-labeled 49 bp oligonucleotide

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Interaction of proliferating cell nuclear antigen with PMS2 is required for MutLα activation and function in mismatch repair

    doi: 10.1073/pnas.1702561114

    Figure Lengend Snippet: PCNA stimulation of MutLα endonuclease activity on linear DNA depends on integrity of the PMS2 721 QRLIAP motif. 50 ng (280 fmol) wild type MutLα, PMS2-Q721A, or PMS2-AAA were incubated with a fluorescently-labeled 49 bp oligonucleotide

    Article Snippet: After blocking with 5% milk for 1 h at room temperature, the membrane was incubated overnight at 4 °C with primary antibody [rabbit anti-PMS2 (sc618), mouse anti-PCNA (sc56), or rabbit anti-MLH1 (sc582); Santa Cruz Biotechnology].

    Techniques: Activity Assay, Incubation, Labeling

    Effect of CXCR2 on hepatocyte proliferation and liver regeneration after I/R injury (A) and partial hepatectomy (B) was determined by immunohistochemical staining for proliferating cell nuclear antigen (PCNA) and quantitative analysis of PCNA labeling. (A) Hepatocyte proliferation in post-ischemic liver (upper panel) after I/R injury showed a significant increase in CXCR2−/− mice. Data are mean ± SEM with n = 3–14 per group. * P

    Journal: PLoS ONE

    Article Title: CXC Chemokines Function as a Rheostat for Hepatocyte Proliferation and Liver Regeneration

    doi: 10.1371/journal.pone.0120092

    Figure Lengend Snippet: Effect of CXCR2 on hepatocyte proliferation and liver regeneration after I/R injury (A) and partial hepatectomy (B) was determined by immunohistochemical staining for proliferating cell nuclear antigen (PCNA) and quantitative analysis of PCNA labeling. (A) Hepatocyte proliferation in post-ischemic liver (upper panel) after I/R injury showed a significant increase in CXCR2−/− mice. Data are mean ± SEM with n = 3–14 per group. * P

    Article Snippet: Proliferating Cell Nuclear Antigen (PCNA) Staining Immunohistochemical staining for PCNA was performed on paraffin-embedded liver tissue with anti-PCNA antibody using DakoCytomation ARK kit (Dako, Copenhagen, Denmark).

    Techniques: Immunohistochemistry, Staining, Labeling, Mouse Assay

    Effects of exogenous MIP-2 and KC treatment on hepatocyte proliferation in the normal liver. Wild-type mice were injected intravenously with low doses of MIP-2 and KC every 24 hours. An identical volume of sterile phosphate-buffered saline (PBS) was used as a vehicle control. Hepatocyte proliferation was determined by immunohistochemical staining for proliferating cell nuclear antigen (PCNA) and quantitative analysis of PCNA labeling. Data are mean ± SEM with n = 4–8 per group. Original magnification was 400X.

    Journal: PLoS ONE

    Article Title: CXC Chemokines Function as a Rheostat for Hepatocyte Proliferation and Liver Regeneration

    doi: 10.1371/journal.pone.0120092

    Figure Lengend Snippet: Effects of exogenous MIP-2 and KC treatment on hepatocyte proliferation in the normal liver. Wild-type mice were injected intravenously with low doses of MIP-2 and KC every 24 hours. An identical volume of sterile phosphate-buffered saline (PBS) was used as a vehicle control. Hepatocyte proliferation was determined by immunohistochemical staining for proliferating cell nuclear antigen (PCNA) and quantitative analysis of PCNA labeling. Data are mean ± SEM with n = 4–8 per group. Original magnification was 400X.

    Article Snippet: Proliferating Cell Nuclear Antigen (PCNA) Staining Immunohistochemical staining for PCNA was performed on paraffin-embedded liver tissue with anti-PCNA antibody using DakoCytomation ARK kit (Dako, Copenhagen, Denmark).

    Techniques: Mouse Assay, Injection, Immunohistochemistry, Staining, Labeling

    Hepatocyte proliferation and liver regeneration in ischemic and non-ischemic liver lobes after I/R injury. (A) Hepatocyte proliferation was determined by immunohistochemical staining for proliferating cell nuclear antigen (PCNA) and quantitative analysis of PCNA labeling. Data are mean ± SEM with n = 3–5 per group. * P

    Journal: PLoS ONE

    Article Title: CXC Chemokines Function as a Rheostat for Hepatocyte Proliferation and Liver Regeneration

    doi: 10.1371/journal.pone.0120092

    Figure Lengend Snippet: Hepatocyte proliferation and liver regeneration in ischemic and non-ischemic liver lobes after I/R injury. (A) Hepatocyte proliferation was determined by immunohistochemical staining for proliferating cell nuclear antigen (PCNA) and quantitative analysis of PCNA labeling. Data are mean ± SEM with n = 3–5 per group. * P

    Article Snippet: Proliferating Cell Nuclear Antigen (PCNA) Staining Immunohistochemical staining for PCNA was performed on paraffin-embedded liver tissue with anti-PCNA antibody using DakoCytomation ARK kit (Dako, Copenhagen, Denmark).

    Techniques: Immunohistochemistry, Staining, Labeling

    Effects of exogenous MIP-2 and KC treatment on hepatocyte proliferation and liver regeneration after partial hepatectomy. Wild-type mice were injected intravenously with high doses or low doses of MIP-2 and KC, starting 24 hours after hepatectomy and continued daily. An identical volume of sterile phosphate-buffered saline (PBS) was used as a vehicle control. (A) Hepatocyte proliferation was determined by immunohistochemical staining for proliferating cell nuclear antigen (PCNA) and quantitative analysis of PCNA labeling. Data are mean ± SEM with n = 4–8 per group. * P

    Journal: PLoS ONE

    Article Title: CXC Chemokines Function as a Rheostat for Hepatocyte Proliferation and Liver Regeneration

    doi: 10.1371/journal.pone.0120092

    Figure Lengend Snippet: Effects of exogenous MIP-2 and KC treatment on hepatocyte proliferation and liver regeneration after partial hepatectomy. Wild-type mice were injected intravenously with high doses or low doses of MIP-2 and KC, starting 24 hours after hepatectomy and continued daily. An identical volume of sterile phosphate-buffered saline (PBS) was used as a vehicle control. (A) Hepatocyte proliferation was determined by immunohistochemical staining for proliferating cell nuclear antigen (PCNA) and quantitative analysis of PCNA labeling. Data are mean ± SEM with n = 4–8 per group. * P

    Article Snippet: Proliferating Cell Nuclear Antigen (PCNA) Staining Immunohistochemical staining for PCNA was performed on paraffin-embedded liver tissue with anti-PCNA antibody using DakoCytomation ARK kit (Dako, Copenhagen, Denmark).

    Techniques: Mouse Assay, Injection, Immunohistochemistry, Staining, Labeling

    Conservation of the zebrafish id1-CRM2 core sequences and its function across evolution. (A) Sequence comparison of zebrafish id1-CRM2-core (Danio) with human (Homo) and mouse (Mus) orthologous sequences. Conserved nucleotides are indicated with an asterisk. Conserved motifs are outlined by yellow boxes comprising putative DNA recognition sequences for the transcription factors FoxA2, Smad (SBM1 and 2), CRE binding protein (CREB), Pknox and EGR1. Nucleotide sequences in green, red or blue correspond to previously identified sequences in the mouse id1 orthologue: Smad binding element (SBE) , a Smad 1/5 binding site and a binding site for an unknown binding protein, respectively. (B-I) Immunohistochemistry of telencephalic transverse sections with antibodies against GFP (B, F), S100ß (C, G) and PCNA (D, H) (merged panels: E, I). (B) Expression of GFP in RGCs at the telencephalic ventricular zone driven by the human id1 regulatory sequences in the zebrafish adult telencephalon. (F) Expression of the human Tg(Hsid1-CRM2) driven reporter construct is up-regulated upon stab wound injury. The injured telencephalic hemisphere is labeled with a white asterisk. (J) Quantification of PCNA and S100ß expression in GFP + cells in the Tg(Hsid1-CRM2) line. (K) Relative population size of type 1 and type 2 RGCs in the control and lesioned hemispheres. The proportion of GFP + /S100β + /PCNA - type 1 and GFP + /S100 + /PCNA + type 2 stem cells is not altered in the injured hemisphere relative to the control hemisphere of the telencephalon. (L, M) Quantification of GFP + cells upon injury. The number of GFP-expressing cells (L) and the intensity ratio between left and right hemispheres comparing undamaged hemisphere (control) and damaged telencephalic hemisphere (M) are both increased following injury. Bars: mean ± SD. Significance is indicated by asterisks: *, .01≤ p

    Journal: bioRxiv

    Article Title: Bone morphogenetic protein signaling regulates Id1 mediated neural stem cell quiescence in the adult zebrafish brain via a phylogenetically conserved enhancer module

    doi: 10.1101/787804

    Figure Lengend Snippet: Conservation of the zebrafish id1-CRM2 core sequences and its function across evolution. (A) Sequence comparison of zebrafish id1-CRM2-core (Danio) with human (Homo) and mouse (Mus) orthologous sequences. Conserved nucleotides are indicated with an asterisk. Conserved motifs are outlined by yellow boxes comprising putative DNA recognition sequences for the transcription factors FoxA2, Smad (SBM1 and 2), CRE binding protein (CREB), Pknox and EGR1. Nucleotide sequences in green, red or blue correspond to previously identified sequences in the mouse id1 orthologue: Smad binding element (SBE) , a Smad 1/5 binding site and a binding site for an unknown binding protein, respectively. (B-I) Immunohistochemistry of telencephalic transverse sections with antibodies against GFP (B, F), S100ß (C, G) and PCNA (D, H) (merged panels: E, I). (B) Expression of GFP in RGCs at the telencephalic ventricular zone driven by the human id1 regulatory sequences in the zebrafish adult telencephalon. (F) Expression of the human Tg(Hsid1-CRM2) driven reporter construct is up-regulated upon stab wound injury. The injured telencephalic hemisphere is labeled with a white asterisk. (J) Quantification of PCNA and S100ß expression in GFP + cells in the Tg(Hsid1-CRM2) line. (K) Relative population size of type 1 and type 2 RGCs in the control and lesioned hemispheres. The proportion of GFP + /S100β + /PCNA - type 1 and GFP + /S100 + /PCNA + type 2 stem cells is not altered in the injured hemisphere relative to the control hemisphere of the telencephalon. (L, M) Quantification of GFP + cells upon injury. The number of GFP-expressing cells (L) and the intensity ratio between left and right hemispheres comparing undamaged hemisphere (control) and damaged telencephalic hemisphere (M) are both increased following injury. Bars: mean ± SD. Significance is indicated by asterisks: *, .01≤ p

    Article Snippet: Primary antibodies used in this study include chicken anti-GFP (1:1000, Aves labs, Davis, CA), mouse anti-PCNA (1:500, Dako, Agilent, Santa Clara, CA) and rabbit anti-S100 (1:400, Dako).

    Techniques: Sequencing, Binding Assay, Immunohistochemistry, Expressing, Construct, Labeling

    The id1 Cis-regulatory module 2 (CRM2) drives ventricular expression and responds with increased expression to stab injury of the adult zebrafish telencephalon. (A) Homology-based search for cis-regulatory modules (CRMs). Schematic representation of the id1 locus with putative CRMs 1-5 highlighted by yellow rectangles. (B-M’) Stable GFP reporter expression of id1 -CRM5 (B and H-H’), id1 -CRM4 (C and I-I’), id1 -CRM3 (D and J-J’), id1 -CRM2 (E and K-K’), id1 -CRM1 (F and L-L’) are shown in comparison with the control TgBAC(id1:GFP) (G and M-M’) for 24 hpf embryos (B-G) and adult telencephala (H-M’). (H-M) GFP reporter expression analyzed in transverse sections from the middle part of the adult telencephalon (location of section schematically indicated in the upper right corner). The Tg(id1-CRM2:GFP) transgenic line (K) recapitulates GFP expression of the TgBAC(id1:GFP) line (M) with strong expression in the ventricular zone (white arrows) and absence of expression in the rostral migratory stream (RMS, white arrowheads). The yellow arrowheads indicate ectopic GFP expression in the tela choroidea (H-H’), cells with appearances of neurons (I-I’ and L-L’) and oligodendrocytes (J-J’). Rectangles in the panels H-M represent the region magnified in H’-M’, respectively. Dashed lines indicate the boundary of the telencephalon. (N-Q) Magnified views of RGCs highlighting type 1 and type 2 RGCs identified by expression of id1-CRM2:GFP (N), S100ß (O) and PCNA (P; all merged in Q). (R-U) Transverse view of a 5 days post injury telencephalon showing the expression of id1-CRM2:GFP (R), S100ß (S) and PCNA (T; all merged in U). The lesion site (left hemisphere) is marked by an asterisk. (V-W) Summary of colocalization analysis of GFP and S100ß expression for BAC and CRM2- id1 constructs. (W) Relative population sizes of type 1 and type 2 RGCs for BAC and id1-CRM2 constructs. (X-Y) Relative population sizes of type 1 and type 2 RGCs (X) and the number of GFP + cells (Y) for id1-CRM2 constructs, comparing lesioned and unlesioned control hemispheres. (Z) id1-CRM2:GFP intensity ratio between left and right hemispheres comparing undamaged telencephalon (control) and damaged telencephalon (lesion). Significance is indicated by asterisks: *, .01≤ p

    Journal: bioRxiv

    Article Title: Bone morphogenetic protein signaling regulates Id1 mediated neural stem cell quiescence in the adult zebrafish brain via a phylogenetically conserved enhancer module

    doi: 10.1101/787804

    Figure Lengend Snippet: The id1 Cis-regulatory module 2 (CRM2) drives ventricular expression and responds with increased expression to stab injury of the adult zebrafish telencephalon. (A) Homology-based search for cis-regulatory modules (CRMs). Schematic representation of the id1 locus with putative CRMs 1-5 highlighted by yellow rectangles. (B-M’) Stable GFP reporter expression of id1 -CRM5 (B and H-H’), id1 -CRM4 (C and I-I’), id1 -CRM3 (D and J-J’), id1 -CRM2 (E and K-K’), id1 -CRM1 (F and L-L’) are shown in comparison with the control TgBAC(id1:GFP) (G and M-M’) for 24 hpf embryos (B-G) and adult telencephala (H-M’). (H-M) GFP reporter expression analyzed in transverse sections from the middle part of the adult telencephalon (location of section schematically indicated in the upper right corner). The Tg(id1-CRM2:GFP) transgenic line (K) recapitulates GFP expression of the TgBAC(id1:GFP) line (M) with strong expression in the ventricular zone (white arrows) and absence of expression in the rostral migratory stream (RMS, white arrowheads). The yellow arrowheads indicate ectopic GFP expression in the tela choroidea (H-H’), cells with appearances of neurons (I-I’ and L-L’) and oligodendrocytes (J-J’). Rectangles in the panels H-M represent the region magnified in H’-M’, respectively. Dashed lines indicate the boundary of the telencephalon. (N-Q) Magnified views of RGCs highlighting type 1 and type 2 RGCs identified by expression of id1-CRM2:GFP (N), S100ß (O) and PCNA (P; all merged in Q). (R-U) Transverse view of a 5 days post injury telencephalon showing the expression of id1-CRM2:GFP (R), S100ß (S) and PCNA (T; all merged in U). The lesion site (left hemisphere) is marked by an asterisk. (V-W) Summary of colocalization analysis of GFP and S100ß expression for BAC and CRM2- id1 constructs. (W) Relative population sizes of type 1 and type 2 RGCs for BAC and id1-CRM2 constructs. (X-Y) Relative population sizes of type 1 and type 2 RGCs (X) and the number of GFP + cells (Y) for id1-CRM2 constructs, comparing lesioned and unlesioned control hemispheres. (Z) id1-CRM2:GFP intensity ratio between left and right hemispheres comparing undamaged telencephalon (control) and damaged telencephalon (lesion). Significance is indicated by asterisks: *, .01≤ p

    Article Snippet: Primary antibodies used in this study include chicken anti-GFP (1:1000, Aves labs, Davis, CA), mouse anti-PCNA (1:500, Dako, Agilent, Santa Clara, CA) and rabbit anti-S100 (1:400, Dako).

    Techniques: Expressing, Transgenic Assay, BAC Assay, Construct

    The conserved BMP response element in the id1-CRM2 is crucial for correct expression of GFP reporter in the ventricular zone and RGCs. (A-C) Scheme showing mutated id1-CRM2 reporter constructs: (A) id1-CRM2 wt construct with putative TF binding sites indicated in grey, (B) id1-CRM2-Δ74 construct which contains a deletion of a 74 bp stretch of the most conserved sequence in id1-CRM2 , (C) id1-CRM2-mut-SBMs construct with mutation in the 2 SBMs (1 and 2) of id1-CRM2. (D) Deletion of the 74 bp stretch in the id1-CRM2 abolished GFP expression in the ventricular zone. (E-H) Enlarged micrographs of (D). (E-H) Immunohistochemistry with GFP (E), S100ß (F) and PCNA (G) antibodies on telencephalic cross-sections of the id1-CRM2-Δ74 transgenic line shows no GFP reporter expression in S100ß+ RGCs (H, merged view). (I-M) Mutations in Smad binding motifs ( SBM1 and 2 ) abolished GFP expression in the ventricular zone. (J-M) Magnification of white-boxed region in (I). (J-M) Immunohistochemistry with GFP (J), S100ß (K) and PCNA (L) antibodies on telencephalic cross-sections of the id1-CRM2-mut-SBMs transgenic line show no colocalization between the RGC marker, S100ß, and GFP (M, merged view). (N) GFP expression driven by id1-CRM2:GFP reporter construct in the ventricular zone (control). (O-P) Immunohistochemistry of telencephalic cross-sections with GFP (O, Q) and PCNA (P). (O, Q) The BRE does not drive GFP expression in the RGCs (O) and is not inducible by telencephalic injury (Q). The left injured side is labelled with a white asterisk. Anteroposterior positions of transverse sections are indicated in the upper right-hand corner of each image. Scale bar = 20 μm (E, F, G, H, J, K, L, M); 200 μm (D, I, N, O, P, Q).

    Journal: bioRxiv

    Article Title: Bone morphogenetic protein signaling regulates Id1 mediated neural stem cell quiescence in the adult zebrafish brain via a phylogenetically conserved enhancer module

    doi: 10.1101/787804

    Figure Lengend Snippet: The conserved BMP response element in the id1-CRM2 is crucial for correct expression of GFP reporter in the ventricular zone and RGCs. (A-C) Scheme showing mutated id1-CRM2 reporter constructs: (A) id1-CRM2 wt construct with putative TF binding sites indicated in grey, (B) id1-CRM2-Δ74 construct which contains a deletion of a 74 bp stretch of the most conserved sequence in id1-CRM2 , (C) id1-CRM2-mut-SBMs construct with mutation in the 2 SBMs (1 and 2) of id1-CRM2. (D) Deletion of the 74 bp stretch in the id1-CRM2 abolished GFP expression in the ventricular zone. (E-H) Enlarged micrographs of (D). (E-H) Immunohistochemistry with GFP (E), S100ß (F) and PCNA (G) antibodies on telencephalic cross-sections of the id1-CRM2-Δ74 transgenic line shows no GFP reporter expression in S100ß+ RGCs (H, merged view). (I-M) Mutations in Smad binding motifs ( SBM1 and 2 ) abolished GFP expression in the ventricular zone. (J-M) Magnification of white-boxed region in (I). (J-M) Immunohistochemistry with GFP (J), S100ß (K) and PCNA (L) antibodies on telencephalic cross-sections of the id1-CRM2-mut-SBMs transgenic line show no colocalization between the RGC marker, S100ß, and GFP (M, merged view). (N) GFP expression driven by id1-CRM2:GFP reporter construct in the ventricular zone (control). (O-P) Immunohistochemistry of telencephalic cross-sections with GFP (O, Q) and PCNA (P). (O, Q) The BRE does not drive GFP expression in the RGCs (O) and is not inducible by telencephalic injury (Q). The left injured side is labelled with a white asterisk. Anteroposterior positions of transverse sections are indicated in the upper right-hand corner of each image. Scale bar = 20 μm (E, F, G, H, J, K, L, M); 200 μm (D, I, N, O, P, Q).

    Article Snippet: Primary antibodies used in this study include chicken anti-GFP (1:1000, Aves labs, Davis, CA), mouse anti-PCNA (1:500, Dako, Agilent, Santa Clara, CA) and rabbit anti-S100 (1:400, Dako).

    Techniques: Expressing, Construct, Binding Assay, Sequencing, Mutagenesis, Immunohistochemistry, Transgenic Assay, Marker

    Deletion mapping of id1-CRM2 identified a 157 bp core region, which confers RGC-specific expression in the adult telencephalon. (A) 5’and 3’ deletions of id1-CRM2 analyzed for expression in zebrafish embryos and adult brains. Results are summarized on the right; + indicates specific GFP expression; +/- and – represent weak and absence of expression, respectively. (B-I) Immunohistochemistry of telencephalic transverse sections with antibodies against GFP (B, F), S100ß (C, G) and PCNA (D, H) (merged panels: E, I). Section levels and areas of magnification are indicated in the upper right-hand corner of the image. (B-E) White arrows show two RGCs. (E) The upper cell is GFP + /S100β + /PCNA - (type 1 RGC) while the lower cell is GFP + /S100 + /PCNA + (type 2 RGC). (F-I) Upon stab wound injury the reporter construct expression is up-regulated. The left injured side is labelled with a white asterisk. (B-I) Section levels and areas of magnification are indicated in the upper right-hand corner of the image. (J) Quantification of PCNA and S100ß expression in id1-CRM2:GFP positive cells. (K) Relative population size of type 1 and type 2 RGCs in the control and lesioned hemisphere. (L, M) Quantification of GFP-positive cells and GFP intensity upon injury. Graphs showing the number of GFP-expressing cells (L) and the intensity ratio between left uninjured (control) and right injured hemispheres respectively (M). Bars: mean ± SD. Significance is indicated by asterisks: *, .01≤ p

    Journal: bioRxiv

    Article Title: Bone morphogenetic protein signaling regulates Id1 mediated neural stem cell quiescence in the adult zebrafish brain via a phylogenetically conserved enhancer module

    doi: 10.1101/787804

    Figure Lengend Snippet: Deletion mapping of id1-CRM2 identified a 157 bp core region, which confers RGC-specific expression in the adult telencephalon. (A) 5’and 3’ deletions of id1-CRM2 analyzed for expression in zebrafish embryos and adult brains. Results are summarized on the right; + indicates specific GFP expression; +/- and – represent weak and absence of expression, respectively. (B-I) Immunohistochemistry of telencephalic transverse sections with antibodies against GFP (B, F), S100ß (C, G) and PCNA (D, H) (merged panels: E, I). Section levels and areas of magnification are indicated in the upper right-hand corner of the image. (B-E) White arrows show two RGCs. (E) The upper cell is GFP + /S100β + /PCNA - (type 1 RGC) while the lower cell is GFP + /S100 + /PCNA + (type 2 RGC). (F-I) Upon stab wound injury the reporter construct expression is up-regulated. The left injured side is labelled with a white asterisk. (B-I) Section levels and areas of magnification are indicated in the upper right-hand corner of the image. (J) Quantification of PCNA and S100ß expression in id1-CRM2:GFP positive cells. (K) Relative population size of type 1 and type 2 RGCs in the control and lesioned hemisphere. (L, M) Quantification of GFP-positive cells and GFP intensity upon injury. Graphs showing the number of GFP-expressing cells (L) and the intensity ratio between left uninjured (control) and right injured hemispheres respectively (M). Bars: mean ± SD. Significance is indicated by asterisks: *, .01≤ p

    Article Snippet: Primary antibodies used in this study include chicken anti-GFP (1:1000, Aves labs, Davis, CA), mouse anti-PCNA (1:500, Dako, Agilent, Santa Clara, CA) and rabbit anti-S100 (1:400, Dako).

    Techniques: Expressing, Immunohistochemistry, Construct

    Generation of a PCNA K164R mutant cell line in RPE-1 using CRISPR/Cas9 A) Schematic of the human PCNA indicating that exon 5 was targeted by CRISPR-Cas9. The K164R mutation was knocked-in utilizing a donor plasmid. B) Schematic of screening PCR and expected PCR product sizes after EcoRI restriction enzyme digestion. C) Representative genotyping PCR. Not targeted (wildtype; 1426 bp), monoallelic knock-in (KIN) ( PCNA KR/- 1E4; 1426 bp, 1168 bp, 258 bp), and biallelic KIN ( PCNA KR/KR 1E12, 2B10; 1168bp, 258 bp). D) Karyotyping analysis from RPE-1 wildtype, PCNA KR/KR (1E12, 2B10) and PCNA KR/- (1E4) cell lines. Blue indicates expec ted RPE-1 karyotype. Red indicates chromosomal abnormalities. E) Western blot analyses of whole cell extracts from wildtype RPE-1, PCNA K164R , and RAD18 -/- cells for MCM2 with α-Tubulin as the loading control. Quantification of MCM2 levels normalized to loading control.

    Journal: bioRxiv

    Article Title: PCNA-K164 ubiquitination facilitates origin licensing and mitotic DNA synthesis

    doi: 10.1101/2020.06.25.172361

    Figure Lengend Snippet: Generation of a PCNA K164R mutant cell line in RPE-1 using CRISPR/Cas9 A) Schematic of the human PCNA indicating that exon 5 was targeted by CRISPR-Cas9. The K164R mutation was knocked-in utilizing a donor plasmid. B) Schematic of screening PCR and expected PCR product sizes after EcoRI restriction enzyme digestion. C) Representative genotyping PCR. Not targeted (wildtype; 1426 bp), monoallelic knock-in (KIN) ( PCNA KR/- 1E4; 1426 bp, 1168 bp, 258 bp), and biallelic KIN ( PCNA KR/KR 1E12, 2B10; 1168bp, 258 bp). D) Karyotyping analysis from RPE-1 wildtype, PCNA KR/KR (1E12, 2B10) and PCNA KR/- (1E4) cell lines. Blue indicates expec ted RPE-1 karyotype. Red indicates chromosomal abnormalities. E) Western blot analyses of whole cell extracts from wildtype RPE-1, PCNA K164R , and RAD18 -/- cells for MCM2 with α-Tubulin as the loading control. Quantification of MCM2 levels normalized to loading control.

    Article Snippet: Primary antibodies were incubated in 5% BLOT-QuickBlocker (G-Biosciences 786-011) as follows: mouse anti-PCNA (Abcam, ab29; 1:3000), rabbit anti-Ubiquityl-PCNA (Lys164) (Cell Signaling, D5C7P, 13439; 1:1000), rabbit anti-RPA32 (S4/8) (Bethyl, A300-245A; 1:2000), rabbit anti-γH2AX (Bethyl, A300-081A; 1:2000), rabbit anti-H2AX (Bethyl, A300-082A; 1:5000), rabbit anti-p-p53 (S15) (Cell Signaling, 9284S; 1:500), mouse anti-p53 (Santa Cruz, sc-126; 1:2000), rabbit anti-p21 (Santa Cruz, sc-397 clone C19; 1:1000), rabbit anti-FANCD2 (Abcam, ab108928; 1:2000), rabbit anti-RAD18 (Bethyl, A300-340A; 1:1000), rabbit anti-MCM2 (Cell Signaling, 4007S; 1:1000; BD Biosciences, 610701; 1:1000), rabbit anti-MCM3 (Cell Signaling, 4012S; 1:1000), rabbit anti-MCM4 (Cell Signaling, 3228S; 1:1000); rabbit anti-MCM7 (Cell Signaling, 3757S; 1:1000), mouse anti-GAPDH (GeneTex, GTX627408; 1:10000), mouse anti-Ku86 (Santa Cruz, B-1, sc-5280; 1:500), mouse anti-α-tubulin (Millipore, T9026, clone DM1A; 1:10000).

    Techniques: Mutagenesis, CRISPR, Plasmid Preparation, Polymerase Chain Reaction, Knock-In, Western Blot

    PCNA K164R mutant cell lines exhibit increased sensitivity to DNA damage A) Chromatin associated PCNA, ubiquityl-PCNA (K164), phospho-RPA32 (S4/8), and γH2AX, with or without 20J/m 2 and 40J/m 2 UV treatment, with histone H2AX as the loading control. Quantification of Ub-PCNA, K164-Ub PCNA, phospho-RPA32, and γH2AX levels normalized to loading control. B) Western blot analyses of whole cell extracts from wildtype RPE-1 and PCNA K164R cells for phospho-p53 (S15), p53, and p21 with or without 10J/m 2 and 40J/m 2 UV treatment, with GAPDH as the loading control. Quantification of phosphor-p53 (S15), p53, and p21 levels normalized to loading control. C) Comparison of drug sensitivity as measured by MTS assay comparing average percent viability in RPE-1 wildtype and PCNA K164R cell lines. Each drug and concentration tested is indicated. Error bars indicate standard deviation and statistical significance was calculated using students t-test with * > .05; ** > .01, *** > .001; n=9 replicate wells across three biological replicates for all data points. D) Representative cell cycle distribution of RPE-1 wildtype and PCNA K164R cell lines treated with or without MMS (10 uM) and MMC (20 nM) for 48 h, based on DNA content (PI). E) Cell cycle distribution of RPE-1 wildtype and PCNA K164R cell lines treated with or without MMS and MMC from three biological replicates. Percent of each population in G1- (green), S- (purple) or G2/M-phase (gray) is shown.

    Journal: bioRxiv

    Article Title: PCNA-K164 ubiquitination facilitates origin licensing and mitotic DNA synthesis

    doi: 10.1101/2020.06.25.172361

    Figure Lengend Snippet: PCNA K164R mutant cell lines exhibit increased sensitivity to DNA damage A) Chromatin associated PCNA, ubiquityl-PCNA (K164), phospho-RPA32 (S4/8), and γH2AX, with or without 20J/m 2 and 40J/m 2 UV treatment, with histone H2AX as the loading control. Quantification of Ub-PCNA, K164-Ub PCNA, phospho-RPA32, and γH2AX levels normalized to loading control. B) Western blot analyses of whole cell extracts from wildtype RPE-1 and PCNA K164R cells for phospho-p53 (S15), p53, and p21 with or without 10J/m 2 and 40J/m 2 UV treatment, with GAPDH as the loading control. Quantification of phosphor-p53 (S15), p53, and p21 levels normalized to loading control. C) Comparison of drug sensitivity as measured by MTS assay comparing average percent viability in RPE-1 wildtype and PCNA K164R cell lines. Each drug and concentration tested is indicated. Error bars indicate standard deviation and statistical significance was calculated using students t-test with * > .05; ** > .01, *** > .001; n=9 replicate wells across three biological replicates for all data points. D) Representative cell cycle distribution of RPE-1 wildtype and PCNA K164R cell lines treated with or without MMS (10 uM) and MMC (20 nM) for 48 h, based on DNA content (PI). E) Cell cycle distribution of RPE-1 wildtype and PCNA K164R cell lines treated with or without MMS and MMC from three biological replicates. Percent of each population in G1- (green), S- (purple) or G2/M-phase (gray) is shown.

    Article Snippet: Primary antibodies were incubated in 5% BLOT-QuickBlocker (G-Biosciences 786-011) as follows: mouse anti-PCNA (Abcam, ab29; 1:3000), rabbit anti-Ubiquityl-PCNA (Lys164) (Cell Signaling, D5C7P, 13439; 1:1000), rabbit anti-RPA32 (S4/8) (Bethyl, A300-245A; 1:2000), rabbit anti-γH2AX (Bethyl, A300-081A; 1:2000), rabbit anti-H2AX (Bethyl, A300-082A; 1:5000), rabbit anti-p-p53 (S15) (Cell Signaling, 9284S; 1:500), mouse anti-p53 (Santa Cruz, sc-126; 1:2000), rabbit anti-p21 (Santa Cruz, sc-397 clone C19; 1:1000), rabbit anti-FANCD2 (Abcam, ab108928; 1:2000), rabbit anti-RAD18 (Bethyl, A300-340A; 1:1000), rabbit anti-MCM2 (Cell Signaling, 4007S; 1:1000; BD Biosciences, 610701; 1:1000), rabbit anti-MCM3 (Cell Signaling, 4012S; 1:1000), rabbit anti-MCM4 (Cell Signaling, 3228S; 1:1000); rabbit anti-MCM7 (Cell Signaling, 3757S; 1:1000), mouse anti-GAPDH (GeneTex, GTX627408; 1:10000), mouse anti-Ku86 (Santa Cruz, B-1, sc-5280; 1:500), mouse anti-α-tubulin (Millipore, T9026, clone DM1A; 1:10000).

    Techniques: Mutagenesis, Western Blot, MTS Assay, Concentration Assay, Standard Deviation

    Phospholipid synthesis activation and AP-1 content in NPcis mice brain as compared to C57BL/6J WT or fos (−/−) KO mice. ( A ) NPcis mice at 4 months of age showing clear PNS tumor burden were treated during 28 days with ASO, SO or vehicle dispensed by an osmotic pump into the caudate putamen (CP) (see Materials and Methods ). After treatment, animals were sacrificed and CP examined by WB for c-Fos and PCNA expression, the latter as an indication of the proliferative status of the tissue. The first two lanes correspond to CP from the left (non-treated) and right (ASO-treated) brain hemispheres from the same NPcis mouse. The 3rd and 4th lanes correspond to the CP from the right, SO-treated or vehicle-treated hemisphere from NPcis mice. The last lane corresponds to CP from a non-treated NPcis mouse. One experiment representative of two performed is shown. Tubulin was stained as a loading control. ( B ) Phospholipid synthesis was measured in vitro in CP from NPcis mice non-treated (1st column), or treated during 28 days as indicated in (A) with SO (2nd column) or from NPcis mice treated only in the right hemisphere with ASO (3rd column, NPcis+ASO) and compared to CP from the left hemisphere of the ASO-treated animals (4th column, NPcis –ASO) or to WT (5th column) or C57BL/6J fos (−/−) KO mice (last column), as indicated. Results are the mean ± SD of two independent experiments performed in triplicate; *p

    Journal: PLoS ONE

    Article Title: Growth of Peripheral and Central Nervous System Tumors Is Supported by Cytoplasmic c-Fos in Humans and Mice

    doi: 10.1371/journal.pone.0009544

    Figure Lengend Snippet: Phospholipid synthesis activation and AP-1 content in NPcis mice brain as compared to C57BL/6J WT or fos (−/−) KO mice. ( A ) NPcis mice at 4 months of age showing clear PNS tumor burden were treated during 28 days with ASO, SO or vehicle dispensed by an osmotic pump into the caudate putamen (CP) (see Materials and Methods ). After treatment, animals were sacrificed and CP examined by WB for c-Fos and PCNA expression, the latter as an indication of the proliferative status of the tissue. The first two lanes correspond to CP from the left (non-treated) and right (ASO-treated) brain hemispheres from the same NPcis mouse. The 3rd and 4th lanes correspond to the CP from the right, SO-treated or vehicle-treated hemisphere from NPcis mice. The last lane corresponds to CP from a non-treated NPcis mouse. One experiment representative of two performed is shown. Tubulin was stained as a loading control. ( B ) Phospholipid synthesis was measured in vitro in CP from NPcis mice non-treated (1st column), or treated during 28 days as indicated in (A) with SO (2nd column) or from NPcis mice treated only in the right hemisphere with ASO (3rd column, NPcis+ASO) and compared to CP from the left hemisphere of the ASO-treated animals (4th column, NPcis –ASO) or to WT (5th column) or C57BL/6J fos (−/−) KO mice (last column), as indicated. Results are the mean ± SD of two independent experiments performed in triplicate; *p

    Article Snippet: Sections were blocked in 4% BSA/0.3% Triton X100 in PBS 10 mM, stained with rabbit anti c-Fos (Santacruz Biotechnology, dilution 1/5000) or rabbit anti PCNA (Santacruz Biotechnology, dilution 1/5000) antibodies, diluted in blocking solution for 48 hours at 4°C and incubated with anti-rabbit biotinilated secondary antibody diluted in blocking solution (Vector, dilution 1/300) for 1h at room temperature.

    Techniques: Activation Assay, Mouse Assay, Allele-specific Oligonucleotide, Western Blot, Expressing, Staining, In Vitro

    Expression and subcellular localization of c-Fos in CNS and PNS tumors from NPcis mice. ( A ) c-Fos expression was determined in mouse brain cortex from a tumor-bearing NPcis mouse (left) and a WT littermate (right) by staining with DAB-peroxidase. Note the abundant c-Fos expression in the NPcis sample, contrasting with the undetectable levels in the WT animal. ( B ) c-Fos (left) and PCNA (right) expression in peripheral nerve tumors stained as in (A). ( C ) Confocal immunofluorescence analysis of peripheral nerve tumors evidencing abundant c-Fos (red) and the ER marker Calnexin (green) immunolabeling. The merged image at right clearly shows c-Fos/ER co-localization. Three additional animals showed the same results as in (A), (B) and (C). Inset: magnification of the box delimited area. Bar: 20 µm.

    Journal: PLoS ONE

    Article Title: Growth of Peripheral and Central Nervous System Tumors Is Supported by Cytoplasmic c-Fos in Humans and Mice

    doi: 10.1371/journal.pone.0009544

    Figure Lengend Snippet: Expression and subcellular localization of c-Fos in CNS and PNS tumors from NPcis mice. ( A ) c-Fos expression was determined in mouse brain cortex from a tumor-bearing NPcis mouse (left) and a WT littermate (right) by staining with DAB-peroxidase. Note the abundant c-Fos expression in the NPcis sample, contrasting with the undetectable levels in the WT animal. ( B ) c-Fos (left) and PCNA (right) expression in peripheral nerve tumors stained as in (A). ( C ) Confocal immunofluorescence analysis of peripheral nerve tumors evidencing abundant c-Fos (red) and the ER marker Calnexin (green) immunolabeling. The merged image at right clearly shows c-Fos/ER co-localization. Three additional animals showed the same results as in (A), (B) and (C). Inset: magnification of the box delimited area. Bar: 20 µm.

    Article Snippet: Sections were blocked in 4% BSA/0.3% Triton X100 in PBS 10 mM, stained with rabbit anti c-Fos (Santacruz Biotechnology, dilution 1/5000) or rabbit anti PCNA (Santacruz Biotechnology, dilution 1/5000) antibodies, diluted in blocking solution for 48 hours at 4°C and incubated with anti-rabbit biotinilated secondary antibody diluted in blocking solution (Vector, dilution 1/300) for 1h at room temperature.

    Techniques: Expressing, Mouse Assay, Staining, Immunofluorescence, Marker, Immunolabeling

    Human brain tumors show abundant c-Fos expression co-localizing with the ER marker calnexin. ( A ) Expression of c-Fos, the ER marker calnexin, and the nuclear marker of proliferating cells PCNA in human brain tumor specimens (n = 156) and non-pathological samples (n = 17). Representative samples of astrocytoma (1st row), GM (2nd row), medulloblastoma (3rd row) and human brain non-pathological samples (4th row) from a tissue array immunostained for c-Fos (red) and calnexin (green); 3rd column is the merge of the previous two columns. Yellow color evidences c-Fos/ER co-localization sites. ( B ) Immunostaining for c-Fos (red), PCNA (green) and the merged image of both is shown for an astrocytoma (1st row), a GM (2nd row) and a medulloblastoma (3rd row). Arrows: proliferating cells with nuclear c-Fos; arrowheads: cells showing predominantly peri-nuclear c-Fos. Bar: 20 µm. Fourth column in A and B is a 20× magnification of the boxed area in the 3rd column.

    Journal: PLoS ONE

    Article Title: Growth of Peripheral and Central Nervous System Tumors Is Supported by Cytoplasmic c-Fos in Humans and Mice

    doi: 10.1371/journal.pone.0009544

    Figure Lengend Snippet: Human brain tumors show abundant c-Fos expression co-localizing with the ER marker calnexin. ( A ) Expression of c-Fos, the ER marker calnexin, and the nuclear marker of proliferating cells PCNA in human brain tumor specimens (n = 156) and non-pathological samples (n = 17). Representative samples of astrocytoma (1st row), GM (2nd row), medulloblastoma (3rd row) and human brain non-pathological samples (4th row) from a tissue array immunostained for c-Fos (red) and calnexin (green); 3rd column is the merge of the previous two columns. Yellow color evidences c-Fos/ER co-localization sites. ( B ) Immunostaining for c-Fos (red), PCNA (green) and the merged image of both is shown for an astrocytoma (1st row), a GM (2nd row) and a medulloblastoma (3rd row). Arrows: proliferating cells with nuclear c-Fos; arrowheads: cells showing predominantly peri-nuclear c-Fos. Bar: 20 µm. Fourth column in A and B is a 20× magnification of the boxed area in the 3rd column.

    Article Snippet: Sections were blocked in 4% BSA/0.3% Triton X100 in PBS 10 mM, stained with rabbit anti c-Fos (Santacruz Biotechnology, dilution 1/5000) or rabbit anti PCNA (Santacruz Biotechnology, dilution 1/5000) antibodies, diluted in blocking solution for 48 hours at 4°C and incubated with anti-rabbit biotinilated secondary antibody diluted in blocking solution (Vector, dilution 1/300) for 1h at room temperature.

    Techniques: Expressing, Marker, Immunostaining

    c-Fos expression is abundant and phospholipid synthesis is activated in a MPNST from a patient with NF1 syndrome. ( A ) Histological examination of a MPNST and a non-pathological specimen excised from an NF1patient, stained with haematoxylin/eosin. Note the neoplastic cells in the MPNST contrasting with the non-pathological (NP) tissue. ( B ) WB for c-Fos and PCNA of the samples shown in (A). Note the lack of c-Fos and PCNA expression in the normal tissue (NP). Tubulin was used as a loading control. ( C ) Phospholipid synthesis capacity in TH from the MPNST and the NP sample. Results are the mean % ± SD of 2 experiments performed in triplicate. TH values from NP were taken as 100%. Phospholipid synthesis activity in the MPNST was > 4-fold that in NP; *p

    Journal: PLoS ONE

    Article Title: Growth of Peripheral and Central Nervous System Tumors Is Supported by Cytoplasmic c-Fos in Humans and Mice

    doi: 10.1371/journal.pone.0009544

    Figure Lengend Snippet: c-Fos expression is abundant and phospholipid synthesis is activated in a MPNST from a patient with NF1 syndrome. ( A ) Histological examination of a MPNST and a non-pathological specimen excised from an NF1patient, stained with haematoxylin/eosin. Note the neoplastic cells in the MPNST contrasting with the non-pathological (NP) tissue. ( B ) WB for c-Fos and PCNA of the samples shown in (A). Note the lack of c-Fos and PCNA expression in the normal tissue (NP). Tubulin was used as a loading control. ( C ) Phospholipid synthesis capacity in TH from the MPNST and the NP sample. Results are the mean % ± SD of 2 experiments performed in triplicate. TH values from NP were taken as 100%. Phospholipid synthesis activity in the MPNST was > 4-fold that in NP; *p

    Article Snippet: Sections were blocked in 4% BSA/0.3% Triton X100 in PBS 10 mM, stained with rabbit anti c-Fos (Santacruz Biotechnology, dilution 1/5000) or rabbit anti PCNA (Santacruz Biotechnology, dilution 1/5000) antibodies, diluted in blocking solution for 48 hours at 4°C and incubated with anti-rabbit biotinilated secondary antibody diluted in blocking solution (Vector, dilution 1/300) for 1h at room temperature.

    Techniques: Expressing, Staining, Western Blot, Activity Assay