Journal: Cell Death & Disease

Article Title: MARVELD1 depletion leads to dysfunction of motor and cognition via regulating glia-dependent neuronal migration during brain development

doi: 10.1038/s41419-018-1027-6

Figure Lengend Snippet: MARVELD1 affected granule cell migration but not proliferation. a Sagittal paraffin-embedded tissue sections of 0- and 6-day-old mice stained with HE. The whole cerebellum with low magnification showed the overall situation of abnormal cells. The arrowheads indicated migrating neurons. b In 6-day-old mice, the width of the EGL and the number of migrating granule cells were analyzed. c Granule cell proliferation and migration were evaluated after a short 1.5-h and a long 30-h chase following BrdU administration in 6-day-old mice in control and MARVELD1 KO animals. The relative cell number was counted. a – c : n = 3 for each genotype. ** p

Article Snippet: For immunohistochemistry and immunofluorescence, the following primary antibodies were used: anti-Calb (1:200, Sigma, no.sab4200543), anti-Blbp (1:100, Abcam, no.ab32423), anti-GFAP (1:200, Abcam, no.ab7260 or ab10062), anti-ITGB1 (1:100, Abcam, no.ab183666 or ab95623), anti-FAK (1:100, Abcam, no.ab40794), anti-p397-FAK (1:100, Abcam, no.ab81298), anti-BrdU (1:100, Sigma, no.b2531), anti-MARVELD1 (1:100, Abcam, no.ab91640 or no.ab169184) and anti-NeuN (1:200, Abcam, no.ab104224).

Techniques: Migration, Mouse Assay, Staining

Journal: Cell Death & Disease

Article Title: MARVELD1 depletion leads to dysfunction of motor and cognition via regulating glia-dependent neuronal migration during brain development

doi: 10.1038/s41419-018-1027-6

Figure Lengend Snippet: MARVELD1 regulated accurate radial migration by affecting the formation of glial fibres. a Immunofluorescence staining of MARVELD1 (green) and glial cells marker GFAP (red) in 6-day-old mice cerebellum. The arrowheads indicated glial cells. b HE staining of 4-week-old GFAP-cre/MARVELD1 fl/fl mice cerebellum sections. The whole cerebellum with low magnification showed the overall situation of abnormal cells location in the molecular layer. n = 3 for each genotype. a and b were different areas from GFAP-cre/MARVELD1 fl/fl cerebellum. c HE staining of 6-day-old control mice and GFAP-cre/MARVELD1 fl/fl mice cerebellum. The whole cerebellum with low magnification showed the overall situation of abnormal cells location in the molecular layer. n = 3 for each genotype. a and b were different areas from control cerebellum. c , d and e are different areas from GFAP-cre/MARVELD1 fl/fl cerebellum. d Granule cell migration was evaluated by a long 60-h chase following BrdU administration in 6-day-old control mice and GFAP-cre/MARVELD1 fl/fl mice. n = 3 for each genotype. e Immunohistochemistry staining with GFAP antibodies in 4-week-old control and GFAP-cre/MARVELD1 fl/fl cerebella. n = 3 for each genotype. a and b were different areas from control cerebellum. c and d were different areas from GFAP-cre/MARVELD1 fl/fl cerebellum. f Immunohistochemistry staining with Calb antibodies in control and GFAP-cre/MARVELD1 fl/fl cerebella in 4-week-old mice. n = 3 for each genotype. a and b are different areas from GFAP-cre/MARVELD1 fl/fl cerebellum. g Sagittal sections of 6-day-old mice cerebellum immunostained with anti-GFAP. GFAP-cre/MARVELD1 fl/fl mice had no obvious glial fibres. n = 3 for each genotype. a and b were different areas from GFAP-cre/MARVELD1 fl/fl cerebellum. h Immunofluorescence of Calb (red) in 6-day-old mice cerebellum. *** p

Article Snippet: For immunohistochemistry and immunofluorescence, the following primary antibodies were used: anti-Calb (1:200, Sigma, no.sab4200543), anti-Blbp (1:100, Abcam, no.ab32423), anti-GFAP (1:200, Abcam, no.ab7260 or ab10062), anti-ITGB1 (1:100, Abcam, no.ab183666 or ab95623), anti-FAK (1:100, Abcam, no.ab40794), anti-p397-FAK (1:100, Abcam, no.ab81298), anti-BrdU (1:100, Sigma, no.b2531), anti-MARVELD1 (1:100, Abcam, no.ab91640 or no.ab169184) and anti-NeuN (1:200, Abcam, no.ab104224).

Techniques: Migration, Immunofluorescence, Staining, Marker, Mouse Assay, Immunohistochemistry

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

Article Title: Tumor suppressor gene Rb is required for self-renewal of spermatogonial stem cells in mice

doi: 10.1073/pnas.1311548110

Figure Lengend Snippet: Rb deficiency blocks self-renewal of GFRa1 + A s spermatogonia. ( A ) Outline of experiment to identify newly generated GFRa1 + A s spermatogonia through BrdU tracing. BrdU marks progeny of cells that were in S phase at time of labeling. Nuclei counterstained with DAPI (blue). ( B ) Mice at P5/P6 or P10/P11 were injected with a single pulse of BrdU. Five days after injection (3–5 d in the P10/P11 experiment), testes were collected for whole-mount seminiferous tubule preparation. Shown are percentages of GFRa1 + A s and A pr spermatogonia that retained BrdU label at time of testis recovery (mean ± SD, n = 3 for each group). * P

Article Snippet: Primary antibodies against active caspase-3 (ab13847, Abcam), BrdU (OBT0030, Accurate Chemical and Scientific), GFRa1 (AF560, R & D Systems), histone H3 (phospho S10, ab5176, Abcam), MVH (AF2030, R & D Systems), PLZF (OP128, EMD Biosciences), and RB (sc-7905, Santa Cruz Biotechnology) were used in the study.

Techniques: Generated, Labeling, Mouse Assay, Injection

Journal: The Journal of Neuroscience

Article Title: Retinoic Acid Signaling Mediates Hair Cell Regeneration by Repressing p27kip and sox2 in Supporting Cells

doi: 10.1523/JNEUROSCI.1099-15.2015

Figure Lengend Snippet: The RA pathway is required for supporting cell proliferation. A–D , To evaluate the role of RA in proliferation, lateral cristae of 4.5-d-old tg(brn3c:mGFP) and tg(brn3c:mGFP;hsp70:dnRARaa-GFP) larvae were laser-ablated and heat-shocked to induce dnRAR-GFP expression after 1 h. A , B , Increase of BrdU-positive cells was detected 48 hpa in treated larvae compared with the nonablated controls. C , D , Again, cell proliferation is induced in ablated lateral crista, and only non-dnRAR cells can enter the cell cycle (arrowheads). E–J , To confirm the results from the inner ear, 4.5-d-old tg(claudinb:GFP) and tg(claudinb:GFP;hsp70:dnRARaa-GFP) larvae were treated with neomycin and heat-shocked to induce dnRAR-GFP expression. E–G , The number of BrdU-positive cells (red) increased significantly 15 h after neomycin-induced hair cell damage in non-dnRAR larvae ( F ). At 40 hpt, cell proliferation was mainly detected in mantle cells ( G ). H–J , Cell proliferation is very low at 15 hpt in dnRAR-overexpressing larvae, and only non-dnRAR cells can enter the cell cycle (non-green cells) ( I ). At 40 hpt, less dnRAR is present and extensive proliferation is found in external cells ( J ).

Article Snippet: Lateral crista: lateral crista hair cells of 4.5-d-old tg(brn3c:mGFP;hsp70:dnraraa-GFP) and tg(brn3c:mGFP) larvae were laser-ablated as previously described, and larvae were heat-shocked at 39°C for 1 h; 24 hpa, larvae were treated with 10 m m BrdU (Sigma-Aldrich) for 24 h, washed two times, and fixed in 4% PFA.

Techniques: Expressing

Journal: Molecular and Cellular Biology

Article Title: A Mammalian Bromodomain Protein, Brd4, Interacts with Replication Factor C and Inhibits Progression to S Phase

doi: 10.1128/MCB.22.18.6509-6520.2002

Figure Lengend Snippet: Brd4 domains required for the interaction with RFC and inhibition of S-phase progression. (A) Diagram of Brd4 deletion constructs. BD, bromodomain; ET, ET domain. Solid boxes represent the presence of indicated domains, and diagonal lines represent the absence of indicated domains. (B) Subcellular localization of GFP-Brd4. NIH 3T3 cells (10 5 ) were transfected with 2 μg of the indicated constructs, and GFP localization was visualized by microscopy 24 h after transfection. (C) HeLa cells synchronized at G 1 were transfected with empty pCMV-Flag vector (Cont, control), pCMV-Flag full-length Brd4, or the indicated deletion constructs and harvested at 16 h. Extracts were precipitated with anti-Flag antibody and analyzed for RFC-140 and Flag-Brd4 by immunoblot analysis. Input represents Flag-Brd4 in 8% of the extracts. (D) Inhibition ofBrdU uptake by ectopically expressed Brd4 deletion constructs. Serum-starved NIH 3T3 cells were transfected with 4 μg of EGFP-Brd4 by Lipofectamine-Plus 4 h prior to release. Cells were allowed to proceed to S phase in the presence of BrdU (10 μM) for 16 h and were fixed and stained for BrdU (diagram at top left). FBS, fetal bovine serum. The number of cells with GFP-Brd4 (green) with or without BrdU incorporation (red) was counted from five independent fields. All cells were detected by Hoechst DNA stain (blue). A total of ∼150 cells was counted in each field. The table at top right indicates the percentage of BrdU-positive cells in the total number of GFP-positive cells. (E) Influence of recombinant Brd4 deletion constructs on the elongation of a singly primed DNA template by the DNA polymerase δ holoenzyme. DNA elongation reactions were carried out with 1 fmol (lanes 1 to 7) and 10 fmol (lanes 8 to 10) of RFC. Thirty-five, 70, 175, and 175 ng of D3 was added to lanes 2, 3, 4 and 9, respectively, and 35, 70, 175, and 175 ng of D1 was added to lanes 5, 6, 7 and 10, respectively. The DNA synthesized in each reaction (in picomoles) was as follows: 9.81 in lane 1, 13.7 in lane 2, 9.82 in lane 3, 9.51 in lane 4, 8.01 in lane 5, 5.01 in lane 6, 2.61 in lane 7, 17.0 in lane 8, 16.4 in lane 9, and 12.2 in lane 10. In the absence of RFC, no DNA synthesis was observed (data not shown).

Article Snippet: Antibodies to PCNA, YY1 and green fluorescent protein (GFP) were obtained from Santa Cruz, Flag M2 was obtained from Sigma, bromodeoxyuridine (BrdU) was obtained from PharMingen, and second antibodies were obtained from Amersham.

Techniques: Inhibition, Construct, Transfection, Microscopy, Plasmid Preparation, Staining, BrdU Incorporation Assay, Recombinant, Synthesized, DNA Synthesis

Journal: Molecular and Cellular Biology

Article Title: A Mammalian Bromodomain Protein, Brd4, Interacts with Replication Factor C and Inhibits Progression to S Phase

doi: 10.1128/MCB.22.18.6509-6520.2002

Figure Lengend Snippet: Cell cycle analysis. (A) Reduced S-phase cells after transient Brd4 transfection. NIH 3T3 cells (2 × 10 6 ) were transfected with 30 μg of GFP-Brd4, Brd4-GFP, or control pEGFP-Cl (GFP only) along with 3 μg of pCMX-IL-2R (left panels). Twenty-four hours after transfection, cells were labeled with BrdU (10 μM) for 30 min and then sorted by anti-IL-2R antibody (GFP + cells, ∼95%). Nuclei were double stained with fluorescein isothiocyanate-conjugated anti-BrdU antibody and PI and analyzed by flow cytometry. Also shown are the results of immunoblot detection of GFP-Brd4 with anti-GFP antibody (right panels). (B) The parental and TET-inducible cells were synchronized by serum starvation, and Brd4 was induced by DOX 24 h prior to release. Cells were harvested at 0 and 16 h after release, stained with PI, and analyzed by flow cytometry. The panels at the right show the percentage of the S-phase population. Values represent the averages from three independent experiments plus standard deviations. Panels at the bottom show the results of immunoblot detection of Brd4 expression.

Article Snippet: Antibodies to PCNA, YY1 and green fluorescent protein (GFP) were obtained from Santa Cruz, Flag M2 was obtained from Sigma, bromodeoxyuridine (BrdU) was obtained from PharMingen, and second antibodies were obtained from Amersham.

Techniques: Cell Cycle Assay, Transfection, Labeling, Staining, Flow Cytometry, Cytometry, Expressing

Journal: bioRxiv

Article Title: Clofarabine commandeers the RNR-α—ZRANB3 nuclear signaling axis

doi: 10.1101/830745

Figure Lengend Snippet: The response to ClF is dependent on RNR-α—independent of its reductase activity. (Relates to Figure S7 – S8 ). A. HEK293T cells were treated with the indicated siRNA(s) for 2 days (note: for single siRNA treatments, an equal amount of siCont was added; for siCont alone treatment, 2X siCont was used), and subsequently, after 30-min treatment with ClF (5 µM) or DMSO, cells were subjected to dual-pulse labeling after which BrdU incorporation in EdU-positive cells was measured. B. HEK293T T-REx™ cells with single integrated copies of RNR-α (C429S)-NLS ( left ), RNR-α-NLS ( middle ), or RNR-α(D57N)-NLS ( right ) were exposed to the specific conditions (tetracycline exposure was for 2 d; ClF concentration was 5 µM; siRNA exposure was 2 days); EdU signal in EdU positive cells was recorded (performed this way because these cells have lower basal DNA synthesis, and are more susceptible to ClF than parent HEK293T cells).

Article Snippet: 1:1000 (WB), 1:200 (IF); 60073-1-Ig (Ms), Proteintech, 1:2000 (WB), 1:200 (IF), 1:300 (PLA for both HeLa and U2OS cells)]; RNR-b [AB57653 (Ms), AbCam, 1:1000 (WB), 1:200 (IF)]; BrdU [NA61, Calbiochem, 1:1000 (Clone MoBu-1, DPA, selective for BrdU over EdU)]; ZRANB3 [A303-033-A (Rb), Bethyl Laboratories.

Techniques: Activity Assay, Labeling, BrdU Incorporation Assay, Concentration Assay, DNA Synthesis

Journal: bioRxiv

Article Title: Clofarabine commandeers the RNR-α—ZRANB3 nuclear signaling axis

doi: 10.1101/830745

Figure Lengend Snippet: A. Representative images for Figure 1F–G . U2OS were treated with either DMSO or ClF (at indicated concentrations) for 30 min and subjected to DPA. Cells were washed with PBS, fixed with methanol, then analyzed by immunofluorescence imaging. Inset: representative images showing orthogonality of the EdU and BrdU detection methods [Click and anti-BrdU (Clone: MoBu-1, which does not detect EdU) antibody detection, respectively], and chromatic orthogonality of the dyes used (red, Cy5; and green, Alexa-488). Scale bar, 10 µm. B. Representative images for Figure 1G . ZRANB3-KO-35 U2OS, ZRANB3-KO-38 U2OS, and ZRANB3-KO-35 U2OS re-expressing ZRANB3 wt , were subjected to treatment and analysis as in A . Scale bar, 10 µm. C. the quantification from B and C fit to the following equation y = {(1-k)/[1+(x/EC50)]} + k, where y is the normalized BrdU intensities in EdU-positive cells, x is the ClF concentration, and k is a constant representing the Y-axis value when [ClF] = ∞. Inset: EC 50 values (µM) derived from the fit. Note: BrdU intensities in each line are normalized relative to that in the DMSO sample for each specific line (hence, all start at 1.0).

Article Snippet: 1:1000 (WB), 1:200 (IF); 60073-1-Ig (Ms), Proteintech, 1:2000 (WB), 1:200 (IF), 1:300 (PLA for both HeLa and U2OS cells)]; RNR-b [AB57653 (Ms), AbCam, 1:1000 (WB), 1:200 (IF)]; BrdU [NA61, Calbiochem, 1:1000 (Clone MoBu-1, DPA, selective for BrdU over EdU)]; ZRANB3 [A303-033-A (Rb), Bethyl Laboratories.

Techniques: Immunofluorescence, Imaging, Expressing, Concentration Assay, Derivative Assay

Journal: bioRxiv

Article Title: Clofarabine commandeers the RNR-α—ZRANB3 nuclear signaling axis

doi: 10.1101/830745

Figure Lengend Snippet: A. HEK293T cells were transfected with the indicated siRNA for 2 days after which time they were treated with DMSO or ClF (5 µM) for 2 h. After this time, dual-pulse imaging was carried out. Quantitation (plot on right) shows changes in basal BrdU incorporation in EdU-positive cells upon knockdown of the specific indicated genes in DMSO-treated cells. B. HEK293T cells were treated with the indicated siRNAs for 2 days and fixed with MeOH, stained with anti-RNR-α- and anti-RNR-β-antibodies, and imaged using a confocal microscope. C. Quantitation of images in B . D–E . HEK293T cells were transfected with the indicated siRNA combination for 2 days and DNA-synthesis rates were assessed by dual-pulse imaging. ( Note 1 : the same total siRNA amount was used for in each case, by balancing single siRNA doses with siCont). [ Note 2 : in D , although siZRANB3 decreases DNA-synthesis in siRNR-α(3)-treated cells, this is minimal compared to the effect on siCont cells].

Article Snippet: 1:1000 (WB), 1:200 (IF); 60073-1-Ig (Ms), Proteintech, 1:2000 (WB), 1:200 (IF), 1:300 (PLA for both HeLa and U2OS cells)]; RNR-b [AB57653 (Ms), AbCam, 1:1000 (WB), 1:200 (IF)]; BrdU [NA61, Calbiochem, 1:1000 (Clone MoBu-1, DPA, selective for BrdU over EdU)]; ZRANB3 [A303-033-A (Rb), Bethyl Laboratories.

Techniques: Transfection, Imaging, Quantitation Assay, BrdU Incorporation Assay, Staining, Microscopy, DNA Synthesis

Journal: bioRxiv

Article Title: Clofarabine commandeers the RNR-α—ZRANB3 nuclear signaling axis

doi: 10.1101/830745

Figure Lengend Snippet: The indicated cell lines ( left panels , HEK293T; right panels , NIH-3T3 wherein the indicated target gene was knocked down using either siRNA or shRNA, respectively) were treated with different doses of the indicated RNR-inhibiting drugs for 2 days. (See Figure 1A and S1A for the chemical structures of the drugs). For HEK293T cells, siRNA treatment occurred 12 h prior to treatment with the compound. After this time, the number of viable cells was measured using AlamarBlue®. Note: nucleoside antimetabolites at these concentrations elicit growth arrest and do not actually kill cells, thus curves do not go to zero. Data were fit to the following equation y = {(1-k)/[1+(x/EC50)]} + k, where y is the normalized BrdU intensities in EdU-positive cells, x is the ClF concentration, and k is a constant representing the Y-axis value when [ClF] = ∞. See also Table 1 .

Article Snippet: 1:1000 (WB), 1:200 (IF); 60073-1-Ig (Ms), Proteintech, 1:2000 (WB), 1:200 (IF), 1:300 (PLA for both HeLa and U2OS cells)]; RNR-b [AB57653 (Ms), AbCam, 1:1000 (WB), 1:200 (IF)]; BrdU [NA61, Calbiochem, 1:1000 (Clone MoBu-1, DPA, selective for BrdU over EdU)]; ZRANB3 [A303-033-A (Rb), Bethyl Laboratories.

Techniques: shRNA, Concentration Assay

Journal: The Journal of Neuroscience

Article Title: Altered Neuron Excitability and Synaptic Plasticity in the Cerebellar Granular Layer of Juvenile Prion Protein Knock-Out Mice with Impaired Motor Control

doi: 10.1523/JNEUROSCI.0409-08.2008

Figure Lengend Snippet: Granule cell proliferation in the germinal zone of the external granular layer. A , Anti-BrdU-stained cerebellum of wild-type and PrP 0/0 mice at different postnatal ages. At P8, a multilayered germinal zone is similarly stained by BrdU both in the EGL

Article Snippet: At different postnatal days (8, 12, 15, 20, 25, 40), PrP0/0 and control C57BL/6J mice were injected intraperitoneally with 5-bromodeoxyuridine (BrdU) (Sigma-Aldrich) (50 μg/g body weight) dissolved in ethanol 25% in PBS.

Techniques: Staining, Mouse Assay

Journal: Stem Cell Research & Therapy

Article Title: PEDF-derived peptide promotes tendon regeneration through its mitogenic effect on tendon stem/progenitor cells

doi: 10.1186/s13287-018-1110-z

Figure Lengend Snippet: The 29-mer induces nucleostemin + TSPC proliferation in an ERK- and STAT3-dependent manner. a BrdU-labeling assay. Primary rabbit tendon cells were cultured to near confluence in culture flasks for 14 days and then verified by nucleostemin immunostaining ( > 98%). Cells were pretreated with PD98059 (10 μM; ERK inhibitor), 50 μM STAT3 inhibitor, or SB203580 (10 μM; p38 MAPK inhibitor) for 2 h before treatment with 10 μM 29-mer and 10 μM BrdU for another 4 h. BrdU (red)-labeled nuclei were detected by immunofluorescence microscopy. Representative images are from three independent experiments. b The percentages of BrdU + cells per total cells (counterstained by Hoechst 33258; blue) were calculated from ten randomly selected microscopic fields in each treatment. Results are expressed as mean ± SD of three independent experiments. * P

Article Snippet: Cells cultured on slides were fixed with 4% paraformaldehyde, treated at 4 °C with methanol for 1 min, and then blocked with 1% goat serum and 5% BSA for 1 h. The cells were stained with antibodies to CD146 (1:50 dilution), nucleostemin (1:100 dilution), or BrdU (1:100 dilution) at room temperature (RT) for 3 h. The slides were subsequently incubated with FITC-donkey anti-rabbit IgG or Alexa Fluor® 647 Goat anti-mouse IgG (1:500 dilution; BioLegend, San Diego, CA) for 20 min and then counterstained with Hoechst 33258 for 6 min.

Techniques: Labeling, Cell Culture, Immunostaining, Immunofluorescence, Microscopy

Journal: Stem Cell Research & Therapy

Article Title: PEDF-derived peptide promotes tendon regeneration through its mitogenic effect on tendon stem/progenitor cells

doi: 10.1186/s13287-018-1110-z

Figure Lengend Snippet: Immunohistochemical analysis of the distribution of various TSPC populations at 1 week postoperation. Tendons were treated with vehicle or 29-mer/alginate gel immediately after surgery. BrdU was injected intraperitoneally on days 0, 3, and 5. Tendons were harvested on day 7. a CD146 immunostaining (brown) and counterstaining with hematoxylin. Representative images are from three independent experiments. HR, healing region. The digital image analysis of CD146 + TSPC was performed blinded on an average of six randomly selected × 400 magnification fields from each section using a Nikon Eclipse 80i microscope equipped with a Leica DC 500 camera. * P

Article Snippet: Cells cultured on slides were fixed with 4% paraformaldehyde, treated at 4 °C with methanol for 1 min, and then blocked with 1% goat serum and 5% BSA for 1 h. The cells were stained with antibodies to CD146 (1:50 dilution), nucleostemin (1:100 dilution), or BrdU (1:100 dilution) at room temperature (RT) for 3 h. The slides were subsequently incubated with FITC-donkey anti-rabbit IgG or Alexa Fluor® 647 Goat anti-mouse IgG (1:500 dilution; BioLegend, San Diego, CA) for 20 min and then counterstained with Hoechst 33258 for 6 min.

Techniques: Immunohistochemistry, Injection, Immunostaining, Microscopy

Journal: Oncogene

Article Title: BRD4 facilitates replication stress-induced DNA damage response

doi: 10.1038/s41388-018-0194-3

Figure Lengend Snippet: Inhibition of BRD4 leads to aberrant DNA replication re-initiation and sensitization to replication stress-inducing agents. A. Schematic of DNA replication re-initiation/restart assay. U2OS cells are first pulse-labeled with BrdU to identify cells in replication phase (S phase), and then treated with HU to induce replication stress. After one hour of HU treatment, HU is removed and cells are replenished with fresh medium with or without BETi (JQ1 or AZD5153). New DNA replication is detected with EdU pulse labeling after a 4-hour recovery. B. Representative images of U2OS in replication re-initiating/restart assays. All cell nuclei are identified with DAPI staining. BrdU labels cells that are in S phase, whereas EdU staining indicates recovered DNA replication activity after HU treatment in these cells. In cells labeled as HU only, HU was not washed-out. Representative images of HU-, DMSO- and JQ1-treated samples are shown. C. Average EdU intensity (per cell) in BrdU positive cells indicates DNA replication re-initiation under various experimental conditions (same as B, JQ1 2 μM, AZD5153 500 nM). All results are normalized to DMSO-treated control group (**** p

Article Snippet: Reagents were purchased from Sigma (St. Louis, MO, USA): Hydroxyurea (H8627), BrdU (B5002); from Selleckchem: JQ1 (S7110), Flavopiridol (S1230), Dinaciclib (S2768), etoposide (S1225), XL413 (S7547), PHA-767491 (S2742).

Techniques: Inhibition, Labeling, Staining, Activity Assay

Journal: Oncogenesis

Article Title: TLR9 re-expression in cancer cells extends the S-phase and stabilizes p16INK4a protein expression

doi: 10.1038/oncsis.2016.49

Figure Lengend Snippet: TLR9 expression affected the cell cycle but not apoptosis. ( a ) Caski cells stably transduced with GFP (left panel) or TLR9 (right panel) encoded pbabe were stained with Annexin V and propidium iodide to determine percentage of apoptotic and necrotic cells. ( b ) HNSCC 136 cells were stably transduced with pbabe (left panel), pbabe -GFP (middle panel) or pbabe-TLR9 (right panel). Cells were pulsed with BrdU for 20 min and cell cycle analysis was performed after BrdU and 7-AAD staining by flow cytometry. ( c ) HNSCC 136 PLVUT'-GFP (left panel) and HNSCC 136 PLVUT'-TLR9 (right panel) were induced with doxycycline and serum deprivated for 2 days. Cells lysate were collected 0, 4, 8 or 24 h after serum addition. Expression of cell cycle proteins were analyzed by western blotting. ( d ) Densitometry analysis of the western blot band from ( c ).

Article Snippet: The cells were then blocked and stained with an anti-BrdU (Biolegend, London, UK) and 7-AAD (Life Technologies).

Techniques: Expressing, Stable Transfection, Transduction, Staining, Cell Cycle Assay, Flow Cytometry, Cytometry, Western Blot

Journal: Nature cell biology

Article Title: Redeployment of Myc and E2f1-3 drives Rb deficient cell cycles

doi: 10.1038/ncb3210

Figure Lengend Snippet: S-G 2 cell cycle arrest in E2f/Myc QKO progenitor cells ( a ) Immunofluorescence (IF) staining of BrdU (green), geminin (green) and P-H3 (red) in crypts from intestines harvested 2 days after induction of Ah-cre expression. Nuclei were stained with DAPI (blue). Data are representative images from n=3 mice per genetic group. ( b ) Quantification of BrdU, geminin and P-H3 staining. Data presented as mean ± s.d., n=3 mice per genetic group. ( c ) Fluorescence-activated cell sorting analysis of cell cycle status in control and E2f/Myc QKO crypts from intestines harvested 2 days after induction of Ah-cre expression. Left, representative histograms from control (n=3 mice) and E2f/Myc QKO (n=5 mice) crypts. Right, quantification of histograms; data presented as mean ± s.d., n=3 (control) or n=5 ( E2f/Myc QKO ) mice. ( d ) IF staining of P-H2AX (red) and IHC staining of cleaved caspase-3 (brown) in crypts. Intestines were harvested 2 days (P-H2AX) or 4 days (cleaved caspase-3) after induction of Ah-cre expression. Data are representative images from n=3 mice per genetic group. ( e ) Quantification of P-H2AX and cleaved caspase-3 staining. Data presented as mean ± s.d., n=3 mice per genetic group. Scale bars in a and d represent 50 μm.

Article Snippet: Antibodies and dilutions used in this study were as follows: BrdU (Dako; M0744, 1:50), geminin (Santa Cruz; sc-13015, 1:100), Serine 10-phosphorylated histone 3 (Millipore; 06-570, 1:250), cleaved caspase-3 (Cell Signaling; 9661, 1:100), phosphorylated H2AX (Cell Signaling; 9718, 1:100), Ccna2 (Santa Cruz; sc-596, 1:250), Cdc2 (Santa Cruz; sc-54, 1:100), Mcm3 (Santa Cruz; sc-9850, 1:500), Pcna (Santa Cruz; sc-56, 1:500), lysozyme (DAKO; A0099, 1:100), Myc (Santa Cruz; sc-764, 1:500), E2f3 (Millipore; 05-551, 1:100) and β-catenin (AbD Serotec; OBT1683, 1:250).

Techniques: Immunofluorescence, Staining, Expressing, Mouse Assay, Fluorescence, FACS, Immunohistochemistry

Journal: Nature

Article Title: mTORC1 mediated translational elongation limits intestinal tumour initiation and growth

doi: 10.1038/nature13896

Figure Lengend Snippet: Wnt-signalling is still active after Raptor deletion and Rapamycin treatment causes regression of established tumours a+b) Representative IHC of MYC and β-catenin showing high MYC levels and nuclear localisation of β-catenin 96hrs after Apc and Apc /Raptor deletion, demonstrating active Wnt-signalling. Nuclear staining (as opposed to membranous staining) of β-catenin is indicative of active Wnt-signalling; Scale bar = 100μm (representative of 3 biological replicates). c) Kaplan-Meyer survival curve of Apc Min /+ mice treated with rapamycin when showing signs of intestinal neoplasia. 10mg/kg rapamycin treatment started when mice showed signs of intestinal disease, and was withdrawn after 30 days. Animals continued to be observed until signs of intestinal neoplasia. Death of animals in the rapamycin group almost always occurred following rapamycin withdrawal. (n=8 per group, ***p-value≤0.001, Log Rank test); d) boxplot showing that 72hr 10mg/kg rapamycin treatment causes an increase in Lysozyme positive cells in tumours. Percent Lysozyme positivity within tumours was calculated using Image J software ( http://imagej.nih.gov/ij/ ). Whiskers are max and min, black line is median (10 tumours from each of 5 mice per group were measured, **p-value≤0.014, Mann-Whitney U test); e) boxplot showing that 72hrs 10mg/kg rapamycin treatment causes a decrease in BrdU positivity within tumours. Percent BrdU positivity within tumours was calculated using Image J software. Whiskers are max and min, black line is median (10 tumours from each of 5 mice per group were measured, **p-value≤0.021, Mann-Whitney U test); f) Representative IHC of Lysozyme, showing a lack of Lysozyme positive paneth cells in remaining cystic tumours after 30 days of 10mg/kg rapamycin treatment; Scale bar = 100μm (representative of 5 biological replicates).

Article Snippet: Antibody concentrations used were as follows: phospho-rpS6Ser235/236 (1:800; Cell Signaling 4858), phospho-4EBP1Thr37/46 (1:500; Cell Signaling 2855), phospho-eEF2Thr56 (1:500; Novus Biologicals NB100-92518), c-MYC (1:200; Santa Cruz sc-764), β-catenin (1:50; BD Biosciences 610154), BrdU (1:200; BD Biosciences 347580), lysozyme (1:150; Dako A099), GFP (1:1,000; Abcam ab6556), p21 (1/4; CNIO Madrid), p16 (1:400; Santa Cruz sc1661), p53 (1/150; Vector Laboratories VPP956).

Techniques: Immunohistochemistry, Staining, Mouse Assay, Software, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Potential benefits of mesenchymal stem cells and electroacupuncture on the trophic factors associated with neurogenesis in mice with ischemic stroke

doi: 10.1038/s41598-018-20481-3

Figure Lengend Snippet: Effect of mBMSC and EA treatment on neurogenesis as detected by BrdU incorporation. ( A ) Photomicrographs and ( B , C ) histograms for BrdU-positive and BrdU/Dcx double-positive cells in the striatum and hippocampus (n = 5). The number of BrdU-positive cells in the striatum was significantly higher in the combined mBMSC and EA treatment group than in the other groups. BrdU/Dcx double-positive cells are indicated by arrows. # p

Article Snippet: They were stained with the following primary antibodies at 4 °C overnight: BrdU (Cat. OBT0030G, AbD Serotec, Oxford, UK), Dcx (Cat. Ab18723, Abcam, Cambridge, UK), Ki67 (Cat. Ab15580, Abcam), NeuN (Cat. MAB377; ABN78, Millipore Corporation, Billerica, MA, USA), mature BDNF (Cat. NB100–98682, Novus Biologicals, LLC, Littleton CO, USA), NT4 (Cat. sc-545, Santa Cruz Biotechnology, Santa Cruz, CA, USA), pCREB (Cat. sc-7978-R, Santa Cruz Biotechnology), PSA-NCAM (Cat. MAB5324, Millipore Corporation), pTrkB (phospho Y515, Cat. ab131483, Abcam), Sox2 (Cat. ab79351, Abcam), and VEGF (Cat. MAB360, Millipore Corporation).

Techniques: BrdU Incorporation Assay

Journal: Genes & Development

Article Title: hMSH3 and hMSH6 interact with PCNA and colocalize with it to replication foci

doi: 10.1101/gad.191201

Figure Lengend Snippet: Colocalization of hMSH6 or hMSH3 and PCNA to replication foci in HeLa cells in vivo. ( A ) Indirect immunofluorescence of HeLa nuclei stained with antibodies against hMSH6 (green) or PCNA (red). Superimposition of the red and green signals gives yellow color, which is indicative of colocalization of the two proteins. ( B ) As in A except that the cells were stained with a polyclonal anti-hMSH3 antibody (green). ( C ) As in A except that cells were labeled with BrdU for 1 h before fixation, and the nuclei then were stained with anti-BrdU antibodies (green). ( D ) As in C except that the nuclei were stained with anti-hMSH3 polyclonal antibody. Note that the MMR protein signals in A and B are green, whereas those in C and D are red.

Article Snippet: The mouse monoclonal antibodies against MSH2 (Ab-2) and PMS2 (Ab-1) were from Calbiochem-Novabiochem, the anti-MLH1 antibody (13271A) was from PharMingen, and the antibodies against PCNA (19F4) and BrdU (5-bromo-2‘-deoxyuridine-5′-monophosphate) conjugated to fluorescein were from Boehringer Mannheim.

Techniques: In Vivo, Immunofluorescence, Staining, Labeling

Journal: The Journal of Experimental Medicine

Article Title: The Transcriptional Repressor Gfi1 Affects Development of Early, Uncommitted c-Kit+ T Cell Progenitors and CD4/CD8 Lineage Decision in the Thymus

doi: 10.1084/jem.20021417

Figure Lengend Snippet: Depletion of c-Kit + cells and alterations of DN subpopulations in Gfi1 −/− mice. (A) Flow cytometric analysis of CD25 and CD44 expression among electronically gated Lin negative (Lin − ) thymocytes. Relative percentages of DN2, DN3, and DN4 cells appear to be altered in Gfi1 −/− mice compared with normal (Gfi1 +/+ ) controls and the emergence of a new population is noted (arrowhead, CD44 hi CD25 int ; n = 10 for each genotype). (B) Thymocytes from WT (Gfi1 +/+ ) or Gfi1 −/− animals were stained for expression of Lin markers CD44 and CD25. Lin − cells and the indicated subsets were analyzed for annexin V binding. The gates for the DN subsets were as indicated in A. The percentage of annexin V + cells for the indicated subsets is given. The analysis reveals that only the DN1 and DN2 subsets of Gfi1 null mice contain significantly more apoptotic cells ( n = 9 for each genotype). The mean values for Gfi1-deficient and WT DN1 cells are 16.8 ± 4.8% and 9.1 ± 3.5%, respectively. This difference is significant at the 0.05 level (P = 0.004). The mean values for Gfi1-deficient and WT DN2 cells are 13.3 ± 3.3% and 6.0 ± 2.9%, respectively. This difference is significant at the 0.05 level (P = 0.001; Student's t test). (C) Lin − thymocytes from WT (Gfi1 +/+ ) or Gfi1 −/− mice were stained for CD44, CD25, and c-Kit expression. The percentage of c-Kit + cells is given for the DN1, DN2, and CD25 int CD44 hi subset in WT and Gfi1 null mice ( n = 5 for each genotype). (D) Lin − thymocytes from WT (Gfi1 +/+ ) or Gfi1 −/− mice were stained for CD44, CD25, and IL-7Rα expression. The percentage of IL-7Rα + cells is given for the DN1, DN2, DN3, DN4, and CD25 int CD44 hi subset in WT and Gfi1 null mice ( n = 4 for each genotype). (E) Three WT and three Gfi1 null mice were analyzed for BrdU incorporation for each time point over a period of 3 d. The indicated cell subsets were analyzed by flow cytometric measurements and the percentages of BrdU + cells were determined. Values are means with standard deviations and are plotted against the time in days (d).

Article Snippet: Three groups of six mice (three WT and three Gfi1−/− for each group) were initially injected intraperitoneally with 1.8 mg/200 μl BrdU (Roth) in saline and then continuously given BrdU at 1 mg/ml in the drinking water that was changed daily.

Techniques: Mouse Assay, Flow Cytometry, Expressing, Staining, Binding Assay, BrdU Incorporation Assay

Journal: The Journal of Experimental Medicine

Article Title: The Transcriptional Repressor Gfi1 Affects Development of Early, Uncommitted c-Kit+ T Cell Progenitors and CD4/CD8 Lineage Decision in the Thymus

doi: 10.1084/jem.20021417

Figure Lengend Snippet: Lack of Gfi1 alters MHC class I–restricted positive selection. (A) Flow cytometric analysis of CD4 and CD8 expression of total or T3.70 + thymocytes from female WT (Gfi1 +/+ ) or Gfi1 −/− mice expressing the HY-TCR transgene. Total thymocyte numbers and the percentages of the respective DN, DP, and SP cells are indicated. Experiments are representative for five different sets of mice. (B) Compilation of percentages of CD8 + /T3.70 + cells from seven female Gfi1 +/+ HY-TCR animals (open bar) and from seven female Gfi1 −/− HY-TCR animals (solid bar). (C) CD69 + /T3.70 + thymocytes from female WT (Gfi1 +/+ ) or Gfi1 −/− mice expressing the HY-TCR transgene. Total thymocyte numbers and the percentages of the respective DN, DP, and SP cells are indicated. Experiments are representative for five different sets of mice. (D) CD8 + or total thymocytes of both HY-TCR transgenic mice and Gfi1 −/− carrying the HY-TCR transgene were analyzed with an antibody against the Vβ8.2 or the Vα3 variable chain of the HY-TCR. The Vα3 chain was detected with the T3.70 clonotypic antibody. (E) Flow cytometric analysis of CD4 and CD8 expression on thymocytes from 8-wk-old male WT (Gfi1 +/+ ) or Gfi1 −/− mice expressing the HY-TCR transgene. Total thymocyte numbers and the percentages of the respective DN, DP, and SP cells are indicated. (F) Three WT and three Gfi1 null mice were analyzed for BrdU incorporation for each time point over a period of 3 d. The indicated cell subsets were analyzed by flow cytometric measurements and the percentages of BrdU + cells were determined. Values are means with standard deviations and are plotted against the time in days (d).

Article Snippet: Three groups of six mice (three WT and three Gfi1−/− for each group) were initially injected intraperitoneally with 1.8 mg/200 μl BrdU (Roth) in saline and then continuously given BrdU at 1 mg/ml in the drinking water that was changed daily.

Techniques: Selection, Flow Cytometry, Expressing, Mouse Assay, Transgenic Assay, BrdU Incorporation Assay

Journal: Oncotarget

Article Title: Ulk1 over-expression in human gastric cancer is correlated with patients' T classification and cancer relapse

doi: 10.18632/oncotarget.16734

Figure Lengend Snippet: Ulk1 shRNA knockdown inhibits gastric cancer cell survival Expressions of Ulk1 protein (A) and Ulk1 mRNA (B) in AGS cells with lentiviral Ulk1 shRNA (“1#” or “2#”) or scramble control shRNA (“shRNA-C”) were shown. Above cells were also subjected to MTT assay (C) , BrdU ELISA assay (D) and Histone DNA ELISA assay (E) to test cell survival, proliferation and apoptosis, respectively. For these assays, exact same number of viable cells with listed shRNA was plated initially (At 0 hour). * p

Article Snippet: BrdU ELISA assay of cell proliferation Cells with applied treatment were incubated with BrdU (10 μM, Cell Signaling Tech, Shanghai, China).

Techniques: shRNA, MTT Assay, Enzyme-linked Immunosorbent Assay

Journal: Oncotarget

Article Title: Ulk1 over-expression in human gastric cancer is correlated with patients' T classification and cancer relapse

doi: 10.18632/oncotarget.16734

Figure Lengend Snippet: Exogenous over-expression of Ulk1 promotes gastric cancer cell survival Expressions of Ulk1 protein (A) and Ulk1 mRNA (B) in AGS cells with exogenous Ulk1 (GFP-tagged) or empty vector (pSuper-puro-GFP, “GFP”) were shown. Above cells were also subjected to MTT assay (C) , BrdU ELISA assay (D) and Histone DNA ELISA assay (E) to test cell survival, proliferation and apoptosis, respectively. For the assays, exact same number of viable cells with Ulk1-GFP or empty vector was plated initially (At 0 hour). * p

Article Snippet: BrdU ELISA assay of cell proliferation Cells with applied treatment were incubated with BrdU (10 μM, Cell Signaling Tech, Shanghai, China).

Techniques: Over Expression, Plasmid Preparation, MTT Assay, Enzyme-linked Immunosorbent Assay