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

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: bioRxiv

Article Title: Regulation of oncogene-induced senescence by the MRE11 and TREX1 nucleases

doi: 10.1101/2023.03.30.534897

Figure Lengend Snippet: ( A ) BJ-RAS V12 cells were grown for 8 days with or without Dox, DMSO and Mirin as indicated. Cells were labeled with two consecutive pulses of IdU and CldU for 20 minutes each and DNA fibers were stretched on glass slides as indicated in Materials and Methods. The length of IdU and CldU tracks, indicative of fork speed, was measured for at least 200 individual ongoing forks (adjacent red and green tracks; see EV ). Results are displayed as super-plots of the distribution of IdU and CldU track length (μm) for 2-3 biological replicates. Median and P-values (two-sided paired-t test) are indicated. ( B ) Mirin suppresses the increased fork asymmetry induced by RAS V12 . Fork asymmetry, which is indicative of increased fork pausing or stalling, was measured as the ratio of the longest to the shortest track for each individual fork in BJ-RAS V12 cells treated with different combinations of Dox, DMSO and Mirin. Increased fork asymmetry in RAS-induced cells, indicative of replication stress, is suppressed by MRE11 inhibition. Median and p-values (non-parametric Mann-Whitney rank sum test) are indicated. A representative experiment is shown (n=2). ( C ) Western blot analysis of DDR factors in BJ cells overexpressing (+Dox) or not (-Dox) RAS v12 and treated or not with 10 µM Mirin. Cytosolic and nuclear fractions were obtained by lysing cells for 5 minutes on ice with a lysis buffer (10 mM Tris pH7.4, 10 mM NaCl, 10 mM MgCl 2 ) containing 0.25% NP-40. Nuclear pellets were lysed in 2x Laemmli buffer and digested with benzonase. ( D ) Mirin prevents the accumulation of micronuclei in BJ-RAS V12 fibroblasts. The frequency of micronuclei was determined in cells treated with various combinations of Dox and Mirin, as indicated in panel A. Mean, SD and p-values (unpaired t-test) are shown for 3 independent experiments.

Article Snippet: Primary antibody mix: Mouse anti-BrdU to detect IdU (1/100, BD 347580), Rat anti-BrdU to detect CldU (1/100, Eurobio clone BU1/75).

Techniques: Labeling, Inhibition, MANN-WHITNEY, Western Blot, Lysis

Journal: bioRxiv

Article Title: Regulation of oncogene-induced senescence by the MRE11 and TREX1 nucleases

doi: 10.1101/2023.03.30.534897

Figure Lengend Snippet: Mirin suppress replication stress in BJ-RAS V12 fibroblasts. ( A ) Experimental scheme. Cells were treated with Dox or Mirin for 8 consecutive days and fork progression was analyzed by DNA fiber spreading. Cells were pulse-labeled with two consecutive pulses of IdU and CldU for 20 minutes each. DNA was counterstained with an anti-ssDNA antibody (blue) and monoclonal antibodies against IdU and CldU (red and green, respectively, see Materials and Methods for details). Ongoing forks appear as adjacent red and green tracks. Total track length is measured as the sum of individual red and green tracks. ( B ) Fork progression in BJ-hTert (n=3) and BJ-RAS v12 cells (n=5) treated or not with Dox. BJ-hTert cells are used here as a control of the effect of Dox alone on fork speed. ( C ) DNA fiber analysis of fork progression in BJ-RAS V12 cells treated or not with Dox for 8 days and treated or not with 10 µM Mirin for the last 3 days of Dox induction. Results are representative of two independent experiments. ( D ) Representative images of DAPI staining in BJ-RAS after induction of RAS v12 for 5 days (Dox at 10 μg/ml) and treated with Mirin or DMSO. Arrow heads point to micronuclei. Scale bar is 20 μm.

Article Snippet: Primary antibody mix: Mouse anti-BrdU to detect IdU (1/100, BD 347580), Rat anti-BrdU to detect CldU (1/100, Eurobio clone BU1/75).

Techniques: Labeling, Bioprocessing, Control, Staining

Journal: The Journal of Cell Biology

Article Title: Cdk2 loss accelerates precursor differentiation and remyelination in the adult central nervous system

doi: 10.1083/jcb.201004146

Figure Lengend Snippet: Cell cycle exit in response to demyelination in the adult brain. (A and B) 7 d after LPC injection, cells were labeled with anti-Olig2, anti-BrdU, and anti-Ki67 in the ASVZ (A) and in the demyelinated lesion (B) of WT (A1–B1) and Cdk2 −/− P90 mice (A2–B2). As BrdU was injected 15 h before sacrifice, triple-immunoreactive Olig2 + /BrdU + /Ki67 + cells measured the proportion of Olig2 + cycling cells while the remaining Olig2 + /BrdU + /Ki67 − cells indicated Olig2 + cells that exited from the cell cycle. Broken lines delineate the different regions. Arrowheads point to double-labeled cells Olig2 + /BrdU + /Ki67 − . cc, corpus callosum; str, striatum; lv, lateral ventricle. Bar, 50 µm. (C) Histogram representing the cell cycle exit index of Olig2 + cells calculated by dividing the number of Olig2 + /BrdU + /Ki67 − cells by the total population of Olig2 + /BrdU + cells. Results are expressed as means ± SEM (error bars; **, P < 0.001) and were analyzed by a t test.

Article Snippet: After several washes in PBS, sections were incubated overnight at 4°C with rat monoclonal anti-BrdU antibody (1:100; AbCys SA).

Techniques: Injection, Labeling

Journal: The Journal of Cell Biology

Article Title: Cdk2 loss accelerates precursor differentiation and remyelination in the adult central nervous system

doi: 10.1083/jcb.201004146

Figure Lengend Snippet: Characterization of adult ASVZ cells in which proliferation is affected by the loss of Cdk2 in response to demyelination. Characterization of proliferating cells in the ASVZ (cc, corpus callosum; str, striatum; lv, lateral ventricle) on sagittal sections obtained from WT (A1–C1) and Cdk2 −/− (A2–C2) P90 mice at 7 dpi. Sections are stained with anti-Ki67 or anti-BrdU and a marker specific for different ASVZ cell types: NG2 (A1–2), Olig2 (B1–2), and Mash1 (C1–2). Broken lines delineate the ASVZ. Arrowheads point to double-labeled cells. Cells in the boxed areas are represented with threefold magnification in the inset. Bar, 50 µm. (A3–C3) Histograms representing the number of proliferating cells of each type in the ASVZ in response to demyelination. Results are expressed as means ± SEM (error bars; *, P < 0.05; **, P < 0.001) and were analyzed by a t test.

Article Snippet: After several washes in PBS, sections were incubated overnight at 4°C with rat monoclonal anti-BrdU antibody (1:100; AbCys SA).

Techniques: Staining, Marker, Labeling