Journal:

Article Title: Local action of the chromatin assembly factor CAF-1 at sites of nucleotide excision repair in vivo

doi: 10.1093/emboj/cdg478

Figure Lengend Snippet: Fig. 2. Recruitment of CAF-1 p60 to replication and damage sites. (A) Characteristic patterns of CAF-1 p60 staining throughout the cell cycle. Cells were pulsed with BrdU followed by double labelling with a polyclonal against p60 (green) and a rat monoclonal against BrdU after denaturation with 4 M HCl (red). (B) CAF-1 p60 is locally recruited to sites of UV damage. Cells were irradiated with 100 J/m2 through filters with either 3 µm or 8 µm pores, with or without post-irradiation incubation, as indicated. Indirect immunofluorescence was performed with a polyclonal against p60 (green) and the anti-thymine dimer mouse monoclonal antibody (red). (C) Damage sites and CAF-1 p60 were visualized as in (B), 30 min after irradiation at the doses indicated. (D) Damage sites and CAF-1 p60 were visualized as in (B), at different times after a dose of 150 J/m2.

Article Snippet: Antibodies Antibodies were used at the following dilutions: anti-thymine dimer mouse monoclonal (Kamiya Biomedical) 1/2000, after denaturation in 0.5 M NaOH for 5 min prior to blocking; anti-XPC rabbit polyclonal 1/1000; anti-HA epitope rat monoclonal (Roche) 1/100; anti-GCN5 (Santa Cruz) 1/200; anti-p300 (Santa Cruz) 1/200; anti-histone H3 acetylated on lysine 9 (Euromedex) 1/200; anti-histone H3 phosphorylated on serine 10 (Upstate Biotechnologies) 1/200; anti-histone H4 acetylated on lysine 5 and anti-histone H4 acetylated on lysine 12 (B.Turner) 1/1000; anti-CAF-1 p60 rabbit polyclonal (made by AgroBio using antigen supplied by D.Roche) or mouse monoclonal (Novus-Abcam) 1/1000; anti-CAF-1 p150 affinity purified rabbit polyclonal (made by AgroBio using antigen supplied by J.-P.Quivy) 1/100 or mouse monoclonal (Novus-Abcam) 1/1000; anti-PCNA mouse monoclonal (DAKO) 1/1000 or rabbit polyclonal (Santa Cruz) 1/20, both after 15 min incubation in 100% methanol at –20°C after PAF fixation; and anti-BrdU rat monoclonal antibody (Becton Dickinson) 1/200.

Techniques: Staining, Irradiation, Incubation, Immunofluorescence

Journal: Stem Cells (Dayton, Ohio)

Article Title: An Endogenous Vitamin K-Dependent Mechanism Regulates Cell Proliferation in the Brain Subventricular Stem Cell Niche

doi: 10.1002/stem.1045

Figure Lengend Snippet: Intracerebroventricular injection of warfarin ((S(−)-3-acetonylbenzyl)-4-hydroxycoumarin)) stimulates cell proliferation in vivo in the regions surrounding the lateral ventricles. Immunostaining of bromodeoxyuridine (BrdU) (in dark, A–D) in brain sections following a single injection of NaCl (1 μl, 9 mg ml −1 ) (A, B) or warfarin (1 μl, 100 mg ml −1 ) (C, D ) in the lateral ventricle of mice. Mice were subjected to intraperitoneal injections of BrdU (50 mg kg −1 ) 68 hours later and sacrificed 72 hours postintracerebroventricular injections. Scale bars = 1 mm (A, C) ; 200 μm (B, D) . In (E) and (F) : BrdU-positive cells were quantified within and in the region (region 2) adjacent to the subventricular zone on sections sampled between 1.18 and 0.14 mm anterior to Bregma, on hemispheres contralateral (contra) or ipsilateral (ipsi) to warfarin or NaCl injection. Means ± SEM numbers of BrdU-positive cells per brain section obtained in regions 1 and 2 from six 4-month-old mice per experimental condition were analyzed with one-way analysis of variance (ANOVA) followed by the post hoc Bonferroni test. *, p < .05; **, p < .01; ***, p < .0001. Double immunostaining for BrdU (green) and glial fibrillary acidic protein (GFAP) (red, G and H) or doublecortin (red, J and K) in ipsilateral brain hemispheres of mice injected with NaCl (G, J) or warfarin (H, K) . Scale bar = 40 μm. Each labeled cell was examined along the z -axis to ensure proper identification of double labeled cells. Percentages of GFAP- (I) or Dcx-over BrdU (L) -positive cells in ipsilateral brain hemispheres of mice injected with NaCl (white bars) or with warfarin (dark bars). Means ± SEM obtained from six 4 months old mice per experimental condition were analyzed with one-way ANOVA followed by the post hoc Bonferroni test. *, p < .05; **, p < .01; ***, p < .0001. Abbreviations: BrdU, bromodeoxyuridine; DCX, doublecortin; GFAP, glial fibrillary acidic protein; ipsi, ipsilateral.

Article Snippet: The cells were then processed for BrdU immunostaining [ ] using a monoclonal rat anti-BrdU antibody (Harlan Sera-Lab, Leicestershire, United Kingdom, http://www.harlanseralab.co.uk).

Techniques: Injection, In Vivo, Immunostaining, Double Immunostaining, Labeling

Journal: Stem Cells (Dayton, Ohio)

Article Title: An Endogenous Vitamin K-Dependent Mechanism Regulates Cell Proliferation in the Brain Subventricular Stem Cell Niche

doi: 10.1002/stem.1045

Figure Lengend Snippet: Regulation of subventricular zone (SVZ) cell culture growth, proliferation, and apoptosis by warfarin ((S(−)-3-acetonylbenzyl)-4-hydroxycoumarin)). Representative photographs of SVZ neurospheres maintained for 5 days either in serum-free medium (SFM) (A) or in SFM supplemented with 1 μg ml −1 warfarin (B) . Scale bar = 200 μm. (C): Growth of SVZ cell cultures maintained for 5 days in either SFM (control) or in SFM supplemented with either 1 μg ml −1 warfarin or with 20 ng ml −1 epidermal growth factor. Percentages of bromodeoxyuridine- (D) or terminal deoxynucleotidyl transferase dUTP nick end labeling-stained (E) nuclei in SVZ cell cultures maintained for 24 hours in SFM (control) or SFM supplemented with 1 μg ml −1 warfarin. (F): Growth of SVZ cell cultures maintained for 5 days in either SFM (control) or SFM supplemented with 1 μg ml −1 warfarin. SVZ cells were derived from newborn or adult rats or mice. Data were obtained from at least three independent experiments each in quadruplicates and are expressed in (C) and (F) as percentages of total viable cell numbers in SFM ± SEM, in (D) and (E) as percentages ± SEM. We used Mann-Whitney test for data statistical analysis. ***, p < .0001; **, p < .01; ns, p = .0570. Abbreviations: BrdU, bromodeoxyuridine; EGF, epidermal growth factor; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.

Article Snippet: The cells were then processed for BrdU immunostaining [ ] using a monoclonal rat anti-BrdU antibody (Harlan Sera-Lab, Leicestershire, United Kingdom, http://www.harlanseralab.co.uk).

Techniques: Cell Culture, Control, TUNEL Assay, Staining, Derivative Assay, MANN-WHITNEY

Journal: Stem Cells (Dayton, Ohio)

Article Title: An Endogenous Vitamin K-Dependent Mechanism Regulates Cell Proliferation in the Brain Subventricular Stem Cell Niche

doi: 10.1002/stem.1045

Figure Lengend Snippet: Gas6 knock out reduces the numbers of subventricular zone (SVZ) cells with stem-like cell properties. (A): Growth of SVZ cell cultures maintained for 5 days in serum-free medium (SFM) (control) or in SFM supplemented with 1 μg ml −1 Gas6. (B): Numbers of primary neurospheres, obtained after incubating SVZ cells derived from wild type (black box) or Gas6 −/− (white box) for 5 days in SFM supplemented with 20 ng ml −1 epidermal growth factor (EGF) are depicted. Neurospheres obtained in each of the conditions were harvested, dissociated as single cells, replated in SFM supplemented with 20 ng ml −1 EGF for 5 additional days and the numbers of secondary neurospheres are also depicted. (C): Numbers of slowly dividing SVZ stem-like cells in wild type or Gas6 −/− mice were obtained by subjecting mice to daily intraperitoneal injections of 50 mg kg −1 bromodeoxyuridine (BrdU) for 5 days and analyzing 24 days later BrdU-positive cells in the SVZ. (D): Numbers of newly generated cells that joined the olfactory bulb in wild-type or Gas6 −/− mice were obtained by subjecting mice to three intraperitoneal injections of 50 mg kg −1 BrdU with a 2-hour interval between each injection during 1 day and analyzing 24 days later, the numbers of BrdU-positive cells within the olfactory bulb. (E): Double immunostaining of BrdU-positive cells with the neuronal marker NeuN in the olfactory bulb, scale bar = 8 μm. (F): Percentages of NeuN (neuronal marker)-positive cells among the BrdU-positive cells in the olfactory bulb in wild-type and Gas6 −/− mice. Data were obtained from at least three experiments (A, B) or six mice (C, D, F) and are depicted as means of percentages of total cell numbers in (A) or numbers of immunoreactive cells per brain section in (C) or density of BrdU-positive cells in (D) or percentages of NeuN-positive cells among BrdU-positive cells in (F) . Mann-Whitney test was used for data statistical analysis; ns, p > .05 for (A) and (F) ; *, p < .05 for (C, D) . Abbreviation: BrdU, bromodeoxyuridine.

Article Snippet: The cells were then processed for BrdU immunostaining [ ] using a monoclonal rat anti-BrdU antibody (Harlan Sera-Lab, Leicestershire, United Kingdom, http://www.harlanseralab.co.uk).

Techniques: Knock-Out, Control, Derivative Assay, Generated, Injection, Double Immunostaining, Marker, MANN-WHITNEY

Journal: Stem Cells (Dayton, Ohio)

Article Title: An Endogenous Vitamin K-Dependent Mechanism Regulates Cell Proliferation in the Brain Subventricular Stem Cell Niche

doi: 10.1002/stem.1045

Figure Lengend Snippet: Protein S inhibits subventricular zone (SVZ) cell proliferation and reverses warfarin ((S(−)-3-acetonylbenzyl)-4-hydroxycoumarin)) effects. (A): Growth of SVZ cell cultures maintained for 5 days in serum-free medium (SFM) (control) or in SFM supplemented with 1 μg ml −1 protein S. (B): Growth of SVZ cell cultures maintained for 5 days in SFM containing 1 μg ml −1 warfarin in absence or presence of 1 μg ml −1 protein S. (C): Growth of SVZ cell cultures maintained for 5 days in SFM containing 10 μg ml −1 vitamin K in the absence or in the presence of 20 μg ml −1 rabbit anti-protein S or an irrelevant rabbit antibody (anti-glial fibrillary acidic protein). (D): Numbers of bromodeoxyuridine (BrdU)-positive cells in the SVZ of 6 months old mice that were subjected to intracerebroventricular injection of anti-protein S or irrelevant antibody (UnAb 1 μl of a 40 μg ml −1 solution). Mice received an intraperitoneal injection of BrdU (50 mg kg −1 ) 68 hours later and were sacrificed 72 hours postintracerebroventricular injections. Data were derived from four mice injected with irrelevant (Un-Ab) antibody and three with anti-protein S. Data were obtained from at least three independent experiments each in quadruplicates and represent means ± SEM of percentages of total cell numbers in (A) , (B), or (C) or of numbers of immunoreactive cells in the SVZ per brain section in (D) . Statistical analysis of the data was performed using the Mann-Whitney test in (A) , (B) , and (D) or the one-way analysis of variance followed by the post hoc Bonferroni test in C . *, p < .05; **, p < .01; ***, p < .001. Abbreviations: PS-Ab, anti-protein S antibody; Un-Ab, unrelated antibody.

Article Snippet: The cells were then processed for BrdU immunostaining [ ] using a monoclonal rat anti-BrdU antibody (Harlan Sera-Lab, Leicestershire, United Kingdom, http://www.harlanseralab.co.uk).

Techniques: Control, Injection, Derivative Assay, MANN-WHITNEY