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    This is a recombinant monoclonal antibody This reformatted mouse antibody was made using the variable domain sequences of the original Mouse IgG format for improved compatibility with existing reagents assays
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    99
    Thermo Fisher anti brdu antibody
    Anti Brdu Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 971 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam rat anti brdu
    Tumors of JPH203-treated mice exhibit an increase in TUNEL-positive nuclei and activation of the amino acid stress response. H E pictures demonstrating PTC/ATC features in ( a ) a vehicle-treated mouse and PTC features in ( b ) a JPH203-treated mouse. c , d <t>Ki67-positive</t> cells and <t>BrdU-positive</t> cells were quantified from each mouse and plotted in the graphs. e TUNEL-positive nuclei were quantified from each mouse and plotted in the graph. f , g Atf5, Slc7a8 and Slc3a2 transcription levels were quantified in vehicle- and JPH203-treated animals. Transcription levels of each gene were normalized to Tuba1a and plotted as fold change relative to vehicle level. All statistical analyses were performed by using a two-tailed Mann-Whitney test
    Rat Anti Brdu, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 3235 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Bio-Rad rat anti brdu
    uPA Deficiency Limits Neoplastic Progression and Phenocopies C5aR1 Deficiency in HPV16 Mice. (A-G) IHC and automated (A-B) or manual (C-G) quantitation of C5aR1 + cells (A), CD45 + leukocytes (B), toluidine blue-stained cells mast cells (C), Ly6G + granulocytes (D), F4/80 + macrophages (E), percentage of <t>BrDU</t> + keratinocytes (F), and <t>CD31</t> + vessels (G) in tissue sections (ear) from HPV16/uPA +/− and HPV16/uPA −/− at 1-, 4-, and 6-months. of age. (H) VEGF and MMP9 levels in ear lysates determined by ELISA from 6-month-old HPV16/uPA +/− and HPV16/uPA −/− ears versus 4-month NT ear skin. Significance determined by unpaired Student’s t test with Welch’s correction. (I) Percentage of ear skin area from indicated genotypes of HPV16 mice exhibiting hyperplasia (hyp; n=6–7 mice per genotype), dysplasia (dys; n=9–14 mice per genotype), and lifetime whole body tumor incidence (HPV16/uPA +/− n=21, HPV16/uPA −/− n=203), with significance determined by Chi-square test, and hazard ratio determined by Kaplan-Meyer analysis. For panels A-H, data points reflect independent mice, and micrographs on right are representative images from 6-month old mice. Panels (A-E, G-H) show epidermal (e), dermal (d) regions, and interface (dotted line). Significance assessed by two-way ANOVA with Bonferroni post-test for multiple comparisons unless otherwise indicated. Data represented as means ± SEM. * p
    Rat Anti Brdu, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 92/100, based on 2103 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson mouse anti brdu
    Wh controls germ cell mitosis exit and meiosis entry. (A-F) Wild-type (A,C,E) and wh 7 mutant (B,D,F) germaria showing <t>1B1</t> (red, fusomes), LamC (red, cap cell nuclear envelopes), <t>BrdU</t> (magenta, S phase marker) and PHH3 (green, M phase marker) in A and B, showing LamC (gray) and CycB (gray, G2/M phase marker) in C and D, and showing DAPI (blue, DNA) and γ-tubulin (red, enriched in the centrosome when cells initiate mitosis) in E and F. (G-M) Wild-type (G,K) and wh 7 mutant (H-J,L,M) germaria showing 1B1 (gray), LamC (gray), C3G (red, synaptonemal complex) and γ-H2AV (green, meiosis-induced double strand break) in G-J, showing LamC (gray) and Orb (gray, oocyte marker) in K-M. Yellow arrows in K and L point to Orb-enriched germ cells (oocytes in wild-type). G′ and I′ show only the C3G channel. (N-P) Wild-type (N) and wh 7 mutant (O,P) egg chambers showing DAPI (gray) and Orb (red). Brackets in A,C, and G mark regions 2a and 2b, where mitosis and meiosis, respectively, occur in wild-type flies. n is the germ cell number in N and O. The white dotted lines in G′ and I′ mark the periphery of germaria. Representative 7-day-old germaria are shown in 3D-reconstructed images. Genotype of wild-type is w 1118 . Scale bars: 10 µm.
    Mouse Anti Brdu, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 2308 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti brdu antibody
    Wh controls germ cell mitosis exit and meiosis entry. (A-F) Wild-type (A,C,E) and wh 7 mutant (B,D,F) germaria showing <t>1B1</t> (red, fusomes), LamC (red, cap cell nuclear envelopes), <t>BrdU</t> (magenta, S phase marker) and PHH3 (green, M phase marker) in A and B, showing LamC (gray) and CycB (gray, G2/M phase marker) in C and D, and showing DAPI (blue, DNA) and γ-tubulin (red, enriched in the centrosome when cells initiate mitosis) in E and F. (G-M) Wild-type (G,K) and wh 7 mutant (H-J,L,M) germaria showing 1B1 (gray), LamC (gray), C3G (red, synaptonemal complex) and γ-H2AV (green, meiosis-induced double strand break) in G-J, showing LamC (gray) and Orb (gray, oocyte marker) in K-M. Yellow arrows in K and L point to Orb-enriched germ cells (oocytes in wild-type). G′ and I′ show only the C3G channel. (N-P) Wild-type (N) and wh 7 mutant (O,P) egg chambers showing DAPI (gray) and Orb (red). Brackets in A,C, and G mark regions 2a and 2b, where mitosis and meiosis, respectively, occur in wild-type flies. n is the germ cell number in N and O. The white dotted lines in G′ and I′ mark the periphery of germaria. Representative 7-day-old germaria are shown in 3D-reconstructed images. Genotype of wild-type is w 1118 . Scale bars: 10 µm.
    Anti Brdu Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1221 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti brdu
    <t>MARVELD1</t> 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 <t>BrdU</t> 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
    Anti Brdu, supplied by Millipore, used in various techniques. Bioz Stars score: 98/100, based on 1133 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson anti brdu antibody
    Selectivity of HIRA for localization to telomeres in ALT cancer cells telomeric DSBs is independent of RPA and is necessary for telomere <t>DNA</t> synthesis. a , Representative IF images of HIRA-YFP localization in ALT + and TEL + cell lines treated with DMSO/PARGi. b , Representative IF images of HIRA-YFP localization in U2OS cells after exposure to 30J/m 2 ultra-violet C (UV-C) and 10 Gy ionizing irradiation (γIR). 5μM PARGi was added for 30 mins following irradiation. c , Left: Western blot validation of RPA70 knockdown in U2OS cells. Middle: Representative IF images of HIRA-YFP localization at telomeres in U2OS cells after RPA70 knockdown. Right: Quantification of HIRA-YFP localization to telomeres in indicated conditions from N = 2 independent assays. d , Western blot validation of HIRA, CABIN1 and UBN1 siRNA knockdown in U2OS cells. e , Graphs of CldU/IdU tract distribution of > 30 telomeric fibers in NT siRNA and HIRA siRNA transfected U2OS-TRF1-FokI cells. f , Representative IF images and quantification of <t>BrdU</t> synthesis at telomeres in indicated cell lines that are transfected with WT-TRF1-FokI and HIRA siRNA. All inhibitor treatments, 5 μM/4hrs. All scale bars in IF panels=5μm. Unless otherwise stated, (n) is the number of cells analyzed and the number of independent assays (N) conducted is represented by black circles. Uncropped blots for c-d .
    Anti Brdu Antibody, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 2802 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore mouse anti brdu
    Selectivity of HIRA for localization to telomeres in ALT cancer cells telomeric DSBs is independent of RPA and is necessary for telomere <t>DNA</t> synthesis. a , Representative IF images of HIRA-YFP localization in ALT + and TEL + cell lines treated with DMSO/PARGi. b , Representative IF images of HIRA-YFP localization in U2OS cells after exposure to 30J/m 2 ultra-violet C (UV-C) and 10 Gy ionizing irradiation (γIR). 5μM PARGi was added for 30 mins following irradiation. c , Left: Western blot validation of RPA70 knockdown in U2OS cells. Middle: Representative IF images of HIRA-YFP localization at telomeres in U2OS cells after RPA70 knockdown. Right: Quantification of HIRA-YFP localization to telomeres in indicated conditions from N = 2 independent assays. d , Western blot validation of HIRA, CABIN1 and UBN1 siRNA knockdown in U2OS cells. e , Graphs of CldU/IdU tract distribution of > 30 telomeric fibers in NT siRNA and HIRA siRNA transfected U2OS-TRF1-FokI cells. f , Representative IF images and quantification of <t>BrdU</t> synthesis at telomeres in indicated cell lines that are transfected with WT-TRF1-FokI and HIRA siRNA. All inhibitor treatments, 5 μM/4hrs. All scale bars in IF panels=5μm. Unless otherwise stated, (n) is the number of cells analyzed and the number of independent assays (N) conducted is represented by black circles. Uncropped blots for c-d .
    Mouse Anti Brdu, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1203 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Agilent technologies mouse anti brdu
    Akt2 deficiency is insufficient to inhibit the development of endometrial carcinoma in <t>Pten</t> +/– mice. ( a ) Histological sections representing the different grades of endometrial neopplasia in Pten +−/− mice (AH, atypical hyperplasia, CIS, carcinoma in situ ). The source of the tissue sections is indicated. ( b ) Incidence of endometrial neoplasia in Pten +/– , Pten +/– Akt2 –/– and wild-type mice. Total number of mice examined in each group is indicated in parentheses. ( c ) <t>BrdU</t> incorporation in the uteri of Pten +/– , Pten +/– Akt2 –/– and wild-type mice. The numbers of mice in each group are indicated in parentheses. P -values are indicated.
    Mouse Anti Brdu, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 92/100, based on 688 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad anti brdu
    Akt2 deficiency is insufficient to inhibit the development of endometrial carcinoma in <t>Pten</t> +/– mice. ( a ) Histological sections representing the different grades of endometrial neopplasia in Pten +−/− mice (AH, atypical hyperplasia, CIS, carcinoma in situ ). The source of the tissue sections is indicated. ( b ) Incidence of endometrial neoplasia in Pten +/– , Pten +/– Akt2 –/– and wild-type mice. Total number of mice examined in each group is indicated in parentheses. ( c ) <t>BrdU</t> incorporation in the uteri of Pten +/– , Pten +/– Akt2 –/– and wild-type mice. The numbers of mice in each group are indicated in parentheses. P -values are indicated.
    Anti Brdu, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 622 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Agilent technologies anti brdu antibody
    Analysis of chondrocyte proliferation in the limbs by <t>BrdU</t> labeling. ( A – D ) <t>Immunohistochemistry</t> of tibiae from BrdU-labeled wild-type ( A ), Runx2 –/– ( B ), Runx2 –/– 3 +/– ( C ), and Runx2 –/– 3 –/– ( D ) embryos at E18.5 using anti-BrdU antibody. The sections were counterstained with toluidine blue. Magnified views of the boxed regions ( a,b,c ) are shown in the same columns. ( A,B ) In wild-type and Runx2 –/– tibiae, the boxed regions a, b , and c represent resting, proliferating, and hypertrophic chondrocytes, respectively. ( C,D ) In the Runx2 –/– 3 +/– and Runx2 –/– 3 –/– tibiae, the boxed regions a, b , and c represent chondrocytes in the epiphyses, metaphyses, and diaphyses, respectively. The growth plates were well formed in wild-type tibiae ( A ) and in Runx2 –/– tibiae ( B ) but not in Runx2 –/– 3 +/– tibiae ( C ) or Runx2 –/– 3 –/– tibiae ( D ). ( Ab – Db ) The columnar alignment of chondrocytes, which is seen in the layer of proliferating chondrocytes, is well formed in the wild-type and Runx2 –/– tibiae, deformed in the Runx2 –/– 3 +/– tibiae, and completely absent in the Runx2 –/– 3 –/– tibiae. ( C,D ) The diaphyses of Runx2 –/– 3 +/– tibiae were composed of slightly enlarged chondrocytes, whereas the entire tibiae of Runx2 –/– 3 –/– embryos were composed of homogeneously small chondrocytes. ( E – I ) Measurement of the frequency of BrdU-positive cells ( E,F ), cell number ( G ), matrix area ( H ), and cell size ( I ) using the tibiae of six wild-type embryos, two Runx3 –/– embryos, six Runx2 –/– embryos, one Runx2 –/– 3 +/– embryo, and two Runx2 –/– 3 –/– embryos. We measured these parameters in each region shown in A – D . ( A,B ) In wild-type and Runx2 –/– tibiae, the regions I, II, and III represent resting, proliferating, and hypertrophic and terminal hypertrophic chondrocytes, respectively. ( C,D ) In the Runx2 –/– 3 +/– and Runx2 –/– 3 –/– tibiae, the regions I, II, and III were arbitrarily determined in the proximal half of the tibiae and represent chondrocytes in the epiphysial, metaphysical, and diaphysial parts of the tibiae, respectively. ( F ) We also counted the number of BrdU-positive cells in femurs, because chondrocyte maturation in Runx2 –/– femurs is more severely inhibited than that in Runx2 –/– ). ( F ) In the measurement of BrdU-positive cells in whole tibiae and femurs, we counted the number of BrdU-positive cells and the total number of cells in regions I and II in wild-type and Runx2 –/– mice and in regions I, II, and III in Runx2 –/– 3 +/– and Runx2 –/– 3 –/– mice, and the mean of the percentage of BrdU-positive cells ± standard deviation (std. dev.) is shown. We measured all of the parameters in three sections of each bone, and the mean ± std. dev. is shown. (*) p
    Anti Brdu Antibody, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 92/100, based on 507 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    GE Healthcare anti brdu antibody
    Analysis of chondrocyte proliferation in the limbs by <t>BrdU</t> labeling. ( A – D ) <t>Immunohistochemistry</t> of tibiae from BrdU-labeled wild-type ( A ), Runx2 –/– ( B ), Runx2 –/– 3 +/– ( C ), and Runx2 –/– 3 –/– ( D ) embryos at E18.5 using anti-BrdU antibody. The sections were counterstained with toluidine blue. Magnified views of the boxed regions ( a,b,c ) are shown in the same columns. ( A,B ) In wild-type and Runx2 –/– tibiae, the boxed regions a, b , and c represent resting, proliferating, and hypertrophic chondrocytes, respectively. ( C,D ) In the Runx2 –/– 3 +/– and Runx2 –/– 3 –/– tibiae, the boxed regions a, b , and c represent chondrocytes in the epiphyses, metaphyses, and diaphyses, respectively. The growth plates were well formed in wild-type tibiae ( A ) and in Runx2 –/– tibiae ( B ) but not in Runx2 –/– 3 +/– tibiae ( C ) or Runx2 –/– 3 –/– tibiae ( D ). ( Ab – Db ) The columnar alignment of chondrocytes, which is seen in the layer of proliferating chondrocytes, is well formed in the wild-type and Runx2 –/– tibiae, deformed in the Runx2 –/– 3 +/– tibiae, and completely absent in the Runx2 –/– 3 –/– tibiae. ( C,D ) The diaphyses of Runx2 –/– 3 +/– tibiae were composed of slightly enlarged chondrocytes, whereas the entire tibiae of Runx2 –/– 3 –/– embryos were composed of homogeneously small chondrocytes. ( E – I ) Measurement of the frequency of BrdU-positive cells ( E,F ), cell number ( G ), matrix area ( H ), and cell size ( I ) using the tibiae of six wild-type embryos, two Runx3 –/– embryos, six Runx2 –/– embryos, one Runx2 –/– 3 +/– embryo, and two Runx2 –/– 3 –/– embryos. We measured these parameters in each region shown in A – D . ( A,B ) In wild-type and Runx2 –/– tibiae, the regions I, II, and III represent resting, proliferating, and hypertrophic and terminal hypertrophic chondrocytes, respectively. ( C,D ) In the Runx2 –/– 3 +/– and Runx2 –/– 3 –/– tibiae, the regions I, II, and III were arbitrarily determined in the proximal half of the tibiae and represent chondrocytes in the epiphysial, metaphysical, and diaphysial parts of the tibiae, respectively. ( F ) We also counted the number of BrdU-positive cells in femurs, because chondrocyte maturation in Runx2 –/– femurs is more severely inhibited than that in Runx2 –/– ). ( F ) In the measurement of BrdU-positive cells in whole tibiae and femurs, we counted the number of BrdU-positive cells and the total number of cells in regions I and II in wild-type and Runx2 –/– mice and in regions I, II, and III in Runx2 –/– 3 +/– and Runx2 –/– 3 –/– mice, and the mean of the percentage of BrdU-positive cells ± standard deviation (std. dev.) is shown. We measured all of the parameters in three sections of each bone, and the mean ± std. dev. is shown. (*) p
    Anti Brdu Antibody, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 356 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    anti brdu antibody - by Bioz Stars, 2021-01
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    N/A
    Conjugation note Unconjugated Application note IHC ICC IF Reactivity note All
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    N/A
    Bromodeoxyuridine 5 bromo 2 deoxyuridine BrdU BUdR BrdUrd is a synthetic nucleoside that is an analog of thymidine It can be incorporated into the newly synthesized DNA of replicating cells
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    Tumors of JPH203-treated mice exhibit an increase in TUNEL-positive nuclei and activation of the amino acid stress response. H E pictures demonstrating PTC/ATC features in ( a ) a vehicle-treated mouse and PTC features in ( b ) a JPH203-treated mouse. c , d Ki67-positive cells and BrdU-positive cells were quantified from each mouse and plotted in the graphs. e TUNEL-positive nuclei were quantified from each mouse and plotted in the graph. f , g Atf5, Slc7a8 and Slc3a2 transcription levels were quantified in vehicle- and JPH203-treated animals. Transcription levels of each gene were normalized to Tuba1a and plotted as fold change relative to vehicle level. All statistical analyses were performed by using a two-tailed Mann-Whitney test

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: The LAT1 inhibitor JPH203 reduces growth of thyroid carcinoma in a fully immunocompetent mouse model

    doi: 10.1186/s13046-018-0907-z

    Figure Lengend Snippet: Tumors of JPH203-treated mice exhibit an increase in TUNEL-positive nuclei and activation of the amino acid stress response. H E pictures demonstrating PTC/ATC features in ( a ) a vehicle-treated mouse and PTC features in ( b ) a JPH203-treated mouse. c , d Ki67-positive cells and BrdU-positive cells were quantified from each mouse and plotted in the graphs. e TUNEL-positive nuclei were quantified from each mouse and plotted in the graph. f , g Atf5, Slc7a8 and Slc3a2 transcription levels were quantified in vehicle- and JPH203-treated animals. Transcription levels of each gene were normalized to Tuba1a and plotted as fold change relative to vehicle level. All statistical analyses were performed by using a two-tailed Mann-Whitney test

    Article Snippet: Primary antibodies anti-Ki67 (Abcam cat. no. ab16667,) and anti-BrdU (Abcam cat. no. ab6326,) were incubated overnight at 4 °C.

    Techniques: Mouse Assay, TUNEL Assay, Activation Assay, Two Tailed Test, MANN-WHITNEY

    uPA Deficiency Limits Neoplastic Progression and Phenocopies C5aR1 Deficiency in HPV16 Mice. (A-G) IHC and automated (A-B) or manual (C-G) quantitation of C5aR1 + cells (A), CD45 + leukocytes (B), toluidine blue-stained cells mast cells (C), Ly6G + granulocytes (D), F4/80 + macrophages (E), percentage of BrDU + keratinocytes (F), and CD31 + vessels (G) in tissue sections (ear) from HPV16/uPA +/− and HPV16/uPA −/− at 1-, 4-, and 6-months. of age. (H) VEGF and MMP9 levels in ear lysates determined by ELISA from 6-month-old HPV16/uPA +/− and HPV16/uPA −/− ears versus 4-month NT ear skin. Significance determined by unpaired Student’s t test with Welch’s correction. (I) Percentage of ear skin area from indicated genotypes of HPV16 mice exhibiting hyperplasia (hyp; n=6–7 mice per genotype), dysplasia (dys; n=9–14 mice per genotype), and lifetime whole body tumor incidence (HPV16/uPA +/− n=21, HPV16/uPA −/− n=203), with significance determined by Chi-square test, and hazard ratio determined by Kaplan-Meyer analysis. For panels A-H, data points reflect independent mice, and micrographs on right are representative images from 6-month old mice. Panels (A-E, G-H) show epidermal (e), dermal (d) regions, and interface (dotted line). Significance assessed by two-way ANOVA with Bonferroni post-test for multiple comparisons unless otherwise indicated. Data represented as means ± SEM. * p

    Journal: Cancer cell

    Article Title: Complement C5a Fosters Squamous Carcinogenesis and Limits T Cell Response to Chemotherapy

    doi: 10.1016/j.ccell.2018.09.003

    Figure Lengend Snippet: uPA Deficiency Limits Neoplastic Progression and Phenocopies C5aR1 Deficiency in HPV16 Mice. (A-G) IHC and automated (A-B) or manual (C-G) quantitation of C5aR1 + cells (A), CD45 + leukocytes (B), toluidine blue-stained cells mast cells (C), Ly6G + granulocytes (D), F4/80 + macrophages (E), percentage of BrDU + keratinocytes (F), and CD31 + vessels (G) in tissue sections (ear) from HPV16/uPA +/− and HPV16/uPA −/− at 1-, 4-, and 6-months. of age. (H) VEGF and MMP9 levels in ear lysates determined by ELISA from 6-month-old HPV16/uPA +/− and HPV16/uPA −/− ears versus 4-month NT ear skin. Significance determined by unpaired Student’s t test with Welch’s correction. (I) Percentage of ear skin area from indicated genotypes of HPV16 mice exhibiting hyperplasia (hyp; n=6–7 mice per genotype), dysplasia (dys; n=9–14 mice per genotype), and lifetime whole body tumor incidence (HPV16/uPA +/− n=21, HPV16/uPA −/− n=203), with significance determined by Chi-square test, and hazard ratio determined by Kaplan-Meyer analysis. For panels A-H, data points reflect independent mice, and micrographs on right are representative images from 6-month old mice. Panels (A-E, G-H) show epidermal (e), dermal (d) regions, and interface (dotted line). Significance assessed by two-way ANOVA with Bonferroni post-test for multiple comparisons unless otherwise indicated. Data represented as means ± SEM. * p

    Article Snippet: Sections were then incubated with blocking buffer containing 5.0% goat serum (Thermo Fisher) and 2.5% bovine serum albumin (BSA), followed by primary antibody incubation, e.g., rat anti-BrdU (MCA2060; Serotec; 1:1000), rat anti-mouse PECAM1/CD31 (MEC 13.3; BioLegend; 1:100), rat anti- Ly6G (1A8; eBiosciences; 1:2000), rat anti-mouse cleaved capsase 3 (#9661; Cell Signaling; 1:200), CD45 (30-F11; BD Pharmingen; 1:500), F4/80 (Cl:A3–1; Serotech; 1:500), C5aR1 (10/92; Abcam; 1:500), or Granzyme B (#NB100–684; Novus Biologicals; 1:200).

    Techniques: Mouse Assay, Immunohistochemistry, Quantitation Assay, Staining, Enzyme-linked Immunosorbent Assay

    C5aR1 Expression Regulates Squamous Carcinogenesis in HPV16 Mice . (A-F) Automated (A) or manual (B-F) quantification of CD45 + cells (A), toluidine blue-stained mast cells (B), Ly6G + granulocytes (C), F4/80 + macrophages (D), CD31 + vessels (E), and percentage of BrdU + keratinocytes (F) in ear skin from HPV16/C5aR1 +/− and HPV16/C5aR1 −/− mice at 1-, 4-, and 6- month of age. Data points in graphs reflect independent mice, and micrographs on right are representative images from 6-month old mice showing epidermal (e), dermal (d) regions, and interface (dotted line). (G) Percentage of ear skin area from indicated genotypes of HPV16 mice exhibiting hyperplasia (hyp; n=8–11), dysplasia (dys; n=8–9), and lifetime whole body SCC incidence (HPV16/C5aR1 +/− n=39 and HPV16/C5aR1 −/− .

    Journal: Cancer cell

    Article Title: Complement C5a Fosters Squamous Carcinogenesis and Limits T Cell Response to Chemotherapy

    doi: 10.1016/j.ccell.2018.09.003

    Figure Lengend Snippet: C5aR1 Expression Regulates Squamous Carcinogenesis in HPV16 Mice . (A-F) Automated (A) or manual (B-F) quantification of CD45 + cells (A), toluidine blue-stained mast cells (B), Ly6G + granulocytes (C), F4/80 + macrophages (D), CD31 + vessels (E), and percentage of BrdU + keratinocytes (F) in ear skin from HPV16/C5aR1 +/− and HPV16/C5aR1 −/− mice at 1-, 4-, and 6- month of age. Data points in graphs reflect independent mice, and micrographs on right are representative images from 6-month old mice showing epidermal (e), dermal (d) regions, and interface (dotted line). (G) Percentage of ear skin area from indicated genotypes of HPV16 mice exhibiting hyperplasia (hyp; n=8–11), dysplasia (dys; n=8–9), and lifetime whole body SCC incidence (HPV16/C5aR1 +/− n=39 and HPV16/C5aR1 −/− .

    Article Snippet: Sections were then incubated with blocking buffer containing 5.0% goat serum (Thermo Fisher) and 2.5% bovine serum albumin (BSA), followed by primary antibody incubation, e.g., rat anti-BrdU (MCA2060; Serotec; 1:1000), rat anti-mouse PECAM1/CD31 (MEC 13.3; BioLegend; 1:100), rat anti- Ly6G (1A8; eBiosciences; 1:2000), rat anti-mouse cleaved capsase 3 (#9661; Cell Signaling; 1:200), CD45 (30-F11; BD Pharmingen; 1:500), F4/80 (Cl:A3–1; Serotech; 1:500), C5aR1 (10/92; Abcam; 1:500), or Granzyme B (#NB100–684; Novus Biologicals; 1:200).

    Techniques: Expressing, Mouse Assay, Staining

    Therapeutic C5aR1 Blockade Sensitizes Established SCCs to Chemotherapy. (A and B) Dosing strategies (top) and PDSC5 growth kinetics (lower graph) in early- (A) or late- (B) stage SCCs. Average tumor volumes reflecting two independent experiments (A; n=12–15) and (B; n=20–24) are shown. (C) Manual quantitation of CD31 + vessels and automated quantitation of cleaved (Clvd) caspase-3 + and BrdU + cells in tissue sections from SCCs shown in panel (B). Each data point shown reflects an independent mouse. (D) Canonical pathway analysis comparing indicated treatment groups using IPA. Gene expression of SCCs generated using the myeloid panel from NanoString. Significant differences (Z-score > |2.0|, dark colors) indicate probability of association of gene expression from NanoString datasets with the indicated canonical pathways. Pathways shaded in yellow were increased in all groups compared to controls (CTL), while the pathway highlighted in pink was increased in PTX and PMX-53 as compared to control. Pathways highlighted in orange, green, and blue were increased in PTX, PMX-53, and PMX-53/PTX as compared to control, respectively. N=9 mice/group. (E) Upstream analysis comparing indicated treatment groups using IPA. Gene expression data from FACS-isolated CD11b + MHCII + F4/80 + macrophages using the pan-cancer immune panel (NanoString). Upstream analyses predict upstream regulators significantly activated or inhibited (Z- score > |2.0|). N = 3–5 mice/group. (F) Dosing strategy (top) and growth kinetics (lower graph) of PDSC5-SCCs in syngeneic mice treated with PTX or PMX-53/PTX, and either isotype control (IgG2b) or αCD8 mAb. Shown are mice pooled from two independent experiments (n=8–12 mice/group). At right is FACS plot showing percentage CD8 + cells (as a percentage of CD3 + .

    Journal: Cancer cell

    Article Title: Complement C5a Fosters Squamous Carcinogenesis and Limits T Cell Response to Chemotherapy

    doi: 10.1016/j.ccell.2018.09.003

    Figure Lengend Snippet: Therapeutic C5aR1 Blockade Sensitizes Established SCCs to Chemotherapy. (A and B) Dosing strategies (top) and PDSC5 growth kinetics (lower graph) in early- (A) or late- (B) stage SCCs. Average tumor volumes reflecting two independent experiments (A; n=12–15) and (B; n=20–24) are shown. (C) Manual quantitation of CD31 + vessels and automated quantitation of cleaved (Clvd) caspase-3 + and BrdU + cells in tissue sections from SCCs shown in panel (B). Each data point shown reflects an independent mouse. (D) Canonical pathway analysis comparing indicated treatment groups using IPA. Gene expression of SCCs generated using the myeloid panel from NanoString. Significant differences (Z-score > |2.0|, dark colors) indicate probability of association of gene expression from NanoString datasets with the indicated canonical pathways. Pathways shaded in yellow were increased in all groups compared to controls (CTL), while the pathway highlighted in pink was increased in PTX and PMX-53 as compared to control. Pathways highlighted in orange, green, and blue were increased in PTX, PMX-53, and PMX-53/PTX as compared to control, respectively. N=9 mice/group. (E) Upstream analysis comparing indicated treatment groups using IPA. Gene expression data from FACS-isolated CD11b + MHCII + F4/80 + macrophages using the pan-cancer immune panel (NanoString). Upstream analyses predict upstream regulators significantly activated or inhibited (Z- score > |2.0|). N = 3–5 mice/group. (F) Dosing strategy (top) and growth kinetics (lower graph) of PDSC5-SCCs in syngeneic mice treated with PTX or PMX-53/PTX, and either isotype control (IgG2b) or αCD8 mAb. Shown are mice pooled from two independent experiments (n=8–12 mice/group). At right is FACS plot showing percentage CD8 + cells (as a percentage of CD3 + .

    Article Snippet: Sections were then incubated with blocking buffer containing 5.0% goat serum (Thermo Fisher) and 2.5% bovine serum albumin (BSA), followed by primary antibody incubation, e.g., rat anti-BrdU (MCA2060; Serotec; 1:1000), rat anti-mouse PECAM1/CD31 (MEC 13.3; BioLegend; 1:100), rat anti- Ly6G (1A8; eBiosciences; 1:2000), rat anti-mouse cleaved capsase 3 (#9661; Cell Signaling; 1:200), CD45 (30-F11; BD Pharmingen; 1:500), F4/80 (Cl:A3–1; Serotech; 1:500), C5aR1 (10/92; Abcam; 1:500), or Granzyme B (#NB100–684; Novus Biologicals; 1:200).

    Techniques: Quantitation Assay, Indirect Immunoperoxidase Assay, Expressing, Generated, CTL Assay, Mouse Assay, FACS, Isolation

    Results of the histological analysis of adult hippocampal neurogenesis in brain sections from WT, TgN3 WT and TgN3 R169C mice after 28 days ( a and c ) or 6 months ( b and d ) under standard (STD), running wheel (RUN) or environmentally enriched (ENR) cage conditions. The percentage of BrdU+/S100β+ ( a and b ) and BrdU+/NeuN+ cells ( c and d ) of all BrdU+ cells was determined to assess effects on the differentiation of BrdU+ cells to astrocytes and neurons. The percentage of BrdU+/S100β+ cells in TgN3 R169C is increased in older mice independent of RUN or ENR ( b ). Data are expressed as mean ± S.E.M.

    Journal: Scientific Reports

    Article Title: Stimulation of adult hippocampal neurogenesis by physical exercise and enriched environment is disturbed in a CADASIL mouse model

    doi: 10.1038/srep45372

    Figure Lengend Snippet: Results of the histological analysis of adult hippocampal neurogenesis in brain sections from WT, TgN3 WT and TgN3 R169C mice after 28 days ( a and c ) or 6 months ( b and d ) under standard (STD), running wheel (RUN) or environmentally enriched (ENR) cage conditions. The percentage of BrdU+/S100β+ ( a and b ) and BrdU+/NeuN+ cells ( c and d ) of all BrdU+ cells was determined to assess effects on the differentiation of BrdU+ cells to astrocytes and neurons. The percentage of BrdU+/S100β+ cells in TgN3 R169C is increased in older mice independent of RUN or ENR ( b ). Data are expressed as mean ± S.E.M.

    Article Snippet: Therefore, a one-in-six series of free-floating brain sections of each animal was pretreated with HCl, followed by an overnight incubation at 4 °C with primary rat anti-BrdU antibody (AbD serotec, 1:500), mouse anti-NeuN (Millipore, 1:1000) and rabbit anti-S100β (Abcam, 1:150).

    Techniques: Mouse Assay

    Results of the histological analysis of adult hippocampal neurogenesis in brain sections from WT, TgN3 WT and TgN3 R169C mice after 28 days ( a , c and e ) or 6 months ( b , d and f ) under standard (STD), running wheel (RUN) or environmentally enriched (ENR) cage conditions. The absolute number of BrdU+ ( a and b ), BrdU+/S100β+ ( c and d ) and BrdU+/NeuN+ cells ( e and f ) was quantified to determine the survival rate of proliferating cells, new astrocytic and new neuronal cells. New neuron survival is reduced in older ( f ) but not younger TgN3 WT mice ( e ). Neurogenic stimulation by RUN or ENR failed in both TgN3 WT and TgN3 R169C independent of the duration ( e and f ). Data are expressed as mean ± S.E.M. *p

    Journal: Scientific Reports

    Article Title: Stimulation of adult hippocampal neurogenesis by physical exercise and enriched environment is disturbed in a CADASIL mouse model

    doi: 10.1038/srep45372

    Figure Lengend Snippet: Results of the histological analysis of adult hippocampal neurogenesis in brain sections from WT, TgN3 WT and TgN3 R169C mice after 28 days ( a , c and e ) or 6 months ( b , d and f ) under standard (STD), running wheel (RUN) or environmentally enriched (ENR) cage conditions. The absolute number of BrdU+ ( a and b ), BrdU+/S100β+ ( c and d ) and BrdU+/NeuN+ cells ( e and f ) was quantified to determine the survival rate of proliferating cells, new astrocytic and new neuronal cells. New neuron survival is reduced in older ( f ) but not younger TgN3 WT mice ( e ). Neurogenic stimulation by RUN or ENR failed in both TgN3 WT and TgN3 R169C independent of the duration ( e and f ). Data are expressed as mean ± S.E.M. *p

    Article Snippet: Therefore, a one-in-six series of free-floating brain sections of each animal was pretreated with HCl, followed by an overnight incubation at 4 °C with primary rat anti-BrdU antibody (AbD serotec, 1:500), mouse anti-NeuN (Millipore, 1:1000) and rabbit anti-S100β (Abcam, 1:150).

    Techniques: Mouse Assay

    Representative confocal images of the triple fluorescent staining of the DG of two different mice ( a – d) : TgN3 WT ENR 6 months; ( e – h ) TgN3 WT STD 28 days). Arrows point to BrdU+ cell nuclei (red, a and e ), NeuN+ cell nuclei (cyan, b and f ), S100β+ cells (green, c and g ), two BrdU+/NeuN+ cell nuclei ( d ) and a BrdU+/S100β+ cell ( h ). Scale bar = 50 μm.

    Journal: Scientific Reports

    Article Title: Stimulation of adult hippocampal neurogenesis by physical exercise and enriched environment is disturbed in a CADASIL mouse model

    doi: 10.1038/srep45372

    Figure Lengend Snippet: Representative confocal images of the triple fluorescent staining of the DG of two different mice ( a – d) : TgN3 WT ENR 6 months; ( e – h ) TgN3 WT STD 28 days). Arrows point to BrdU+ cell nuclei (red, a and e ), NeuN+ cell nuclei (cyan, b and f ), S100β+ cells (green, c and g ), two BrdU+/NeuN+ cell nuclei ( d ) and a BrdU+/S100β+ cell ( h ). Scale bar = 50 μm.

    Article Snippet: Therefore, a one-in-six series of free-floating brain sections of each animal was pretreated with HCl, followed by an overnight incubation at 4 °C with primary rat anti-BrdU antibody (AbD serotec, 1:500), mouse anti-NeuN (Millipore, 1:1000) and rabbit anti-S100β (Abcam, 1:150).

    Techniques: Staining, Mouse Assay

    Wh controls germ cell mitosis exit and meiosis entry. (A-F) Wild-type (A,C,E) and wh 7 mutant (B,D,F) germaria showing 1B1 (red, fusomes), LamC (red, cap cell nuclear envelopes), BrdU (magenta, S phase marker) and PHH3 (green, M phase marker) in A and B, showing LamC (gray) and CycB (gray, G2/M phase marker) in C and D, and showing DAPI (blue, DNA) and γ-tubulin (red, enriched in the centrosome when cells initiate mitosis) in E and F. (G-M) Wild-type (G,K) and wh 7 mutant (H-J,L,M) germaria showing 1B1 (gray), LamC (gray), C3G (red, synaptonemal complex) and γ-H2AV (green, meiosis-induced double strand break) in G-J, showing LamC (gray) and Orb (gray, oocyte marker) in K-M. Yellow arrows in K and L point to Orb-enriched germ cells (oocytes in wild-type). G′ and I′ show only the C3G channel. (N-P) Wild-type (N) and wh 7 mutant (O,P) egg chambers showing DAPI (gray) and Orb (red). Brackets in A,C, and G mark regions 2a and 2b, where mitosis and meiosis, respectively, occur in wild-type flies. n is the germ cell number in N and O. The white dotted lines in G′ and I′ mark the periphery of germaria. Representative 7-day-old germaria are shown in 3D-reconstructed images. Genotype of wild-type is w 1118 . Scale bars: 10 µm.

    Journal: Development (Cambridge, England)

    Article Title: WD40 protein Wuho controls germline homeostasis via TRIM-NHL tumor suppressor Mei-p26 in Drosophila

    doi: 10.1242/dev.182063

    Figure Lengend Snippet: Wh controls germ cell mitosis exit and meiosis entry. (A-F) Wild-type (A,C,E) and wh 7 mutant (B,D,F) germaria showing 1B1 (red, fusomes), LamC (red, cap cell nuclear envelopes), BrdU (magenta, S phase marker) and PHH3 (green, M phase marker) in A and B, showing LamC (gray) and CycB (gray, G2/M phase marker) in C and D, and showing DAPI (blue, DNA) and γ-tubulin (red, enriched in the centrosome when cells initiate mitosis) in E and F. (G-M) Wild-type (G,K) and wh 7 mutant (H-J,L,M) germaria showing 1B1 (gray), LamC (gray), C3G (red, synaptonemal complex) and γ-H2AV (green, meiosis-induced double strand break) in G-J, showing LamC (gray) and Orb (gray, oocyte marker) in K-M. Yellow arrows in K and L point to Orb-enriched germ cells (oocytes in wild-type). G′ and I′ show only the C3G channel. (N-P) Wild-type (N) and wh 7 mutant (O,P) egg chambers showing DAPI (gray) and Orb (red). Brackets in A,C, and G mark regions 2a and 2b, where mitosis and meiosis, respectively, occur in wild-type flies. n is the germ cell number in N and O. The white dotted lines in G′ and I′ mark the periphery of germaria. Representative 7-day-old germaria are shown in 3D-reconstructed images. Genotype of wild-type is w 1118 . Scale bars: 10 µm.

    Article Snippet: The following primary antibodies were used at the indicated dilutions in blocking buffer (Goal Bio, GBW-3400): mouse anti-1B1 [1:30, Developmental Studies Hybridoma Bank (DSHB), 7H9 1B1], mouse anti-LamC (1:40, DSHB, LC28.26), mouse anti-BrdU (1:20, BD Biosciences, clone B44), mouse anti-Fasciclin III (1:40, DSHB, 7G10), rabbit anti-Vasa (1:250, Santa Cruz Biotechnology, sc-30210), mouse anti-C(3)G (1:500, a gift from R.S.

    Techniques: Mutagenesis, Marker

    Wh controls maintenance and abscission of GSCs and synchronous division of GSC progeny in a cell-autonomous manner. (A,B) Wild-type (WT) (A) and wh 7 mutant (B) germaria with 1B1 (gray, fusomes), LamC (gray, terminal filament and cap cell nuclear envelopes), BrdU (gray, S phase marker) and DAPI (blue, DNA). Insets in A are the enlarged view from different focal planes of the 16-cell cyst marked by a dotted line. Arabic numerals show germ cell number in the 16-cell cyst shown in A, and the stem-cyst in B. Note that the stem-cyst in B has 20 germ cells, whereas only one germ cell is positive for BrdU labeling (red arrow), indicating asynchronous division. (C,D) Wild-type (C) and wh 7 mutant (D) germaria showing 1B1 (fusome), LamC (red), phospho-tyrosine [PY] (yellow, ring canals) and DAPI (gray). (E-F′) Wild-type (E) and wh 7 mutant (F) 8-cell cysts showing 1B1 (red), BrdU (red) and DAPI (gray). E′ and F′ show the BrdU channel. (G) Number of GSCs or stem-cysts per germaria in 1-, 7- and 14-day-old (D1, D7 and D14, respectively) flies of indicated genotypes. (H) The percentage of germaria carrying stem-cyst(s) in flies with indicated genotypes. (I) Percentage of germaria with the largest cyst containing indicated germ cell numbers. (J,K) nos > gfp (J) and nos > wh RNAi germaria (K) showing 1B1 (gray), LamC (gray) and Wh (red). (L,M) nos > gfp (L) and nos > wh RNAi germaria (M) showing 1B1 (gray), and LamC (gray). (N) Percentage of germaria carrying indicated number of GSCs in flies with indicated genotypes. GSCs are outlined by white solid lines; stem-cysts are outlined by yellow dotted lines; GSC progeny are outlined by white dotted lines. Data in G and H are mean±s.e.m. The stem-cysts in H are compared using two-tailed Student's t -test. Numbers of cyst cells (I) and GSCs (N) were compared using a chi-squared test. The numbers above the bars in G,I and N are numbers of analyzed germaria. *** P

    Journal: Development (Cambridge, England)

    Article Title: WD40 protein Wuho controls germline homeostasis via TRIM-NHL tumor suppressor Mei-p26 in Drosophila

    doi: 10.1242/dev.182063

    Figure Lengend Snippet: Wh controls maintenance and abscission of GSCs and synchronous division of GSC progeny in a cell-autonomous manner. (A,B) Wild-type (WT) (A) and wh 7 mutant (B) germaria with 1B1 (gray, fusomes), LamC (gray, terminal filament and cap cell nuclear envelopes), BrdU (gray, S phase marker) and DAPI (blue, DNA). Insets in A are the enlarged view from different focal planes of the 16-cell cyst marked by a dotted line. Arabic numerals show germ cell number in the 16-cell cyst shown in A, and the stem-cyst in B. Note that the stem-cyst in B has 20 germ cells, whereas only one germ cell is positive for BrdU labeling (red arrow), indicating asynchronous division. (C,D) Wild-type (C) and wh 7 mutant (D) germaria showing 1B1 (fusome), LamC (red), phospho-tyrosine [PY] (yellow, ring canals) and DAPI (gray). (E-F′) Wild-type (E) and wh 7 mutant (F) 8-cell cysts showing 1B1 (red), BrdU (red) and DAPI (gray). E′ and F′ show the BrdU channel. (G) Number of GSCs or stem-cysts per germaria in 1-, 7- and 14-day-old (D1, D7 and D14, respectively) flies of indicated genotypes. (H) The percentage of germaria carrying stem-cyst(s) in flies with indicated genotypes. (I) Percentage of germaria with the largest cyst containing indicated germ cell numbers. (J,K) nos > gfp (J) and nos > wh RNAi germaria (K) showing 1B1 (gray), LamC (gray) and Wh (red). (L,M) nos > gfp (L) and nos > wh RNAi germaria (M) showing 1B1 (gray), and LamC (gray). (N) Percentage of germaria carrying indicated number of GSCs in flies with indicated genotypes. GSCs are outlined by white solid lines; stem-cysts are outlined by yellow dotted lines; GSC progeny are outlined by white dotted lines. Data in G and H are mean±s.e.m. The stem-cysts in H are compared using two-tailed Student's t -test. Numbers of cyst cells (I) and GSCs (N) were compared using a chi-squared test. The numbers above the bars in G,I and N are numbers of analyzed germaria. *** P

    Article Snippet: The following primary antibodies were used at the indicated dilutions in blocking buffer (Goal Bio, GBW-3400): mouse anti-1B1 [1:30, Developmental Studies Hybridoma Bank (DSHB), 7H9 1B1], mouse anti-LamC (1:40, DSHB, LC28.26), mouse anti-BrdU (1:20, BD Biosciences, clone B44), mouse anti-Fasciclin III (1:40, DSHB, 7G10), rabbit anti-Vasa (1:250, Santa Cruz Biotechnology, sc-30210), mouse anti-C(3)G (1:500, a gift from R.S.

    Techniques: Mutagenesis, Marker, Labeling, Two Tailed Test

    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

    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

    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

    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

    Selectivity of HIRA for localization to telomeres in ALT cancer cells telomeric DSBs is independent of RPA and is necessary for telomere DNA synthesis. a , Representative IF images of HIRA-YFP localization in ALT + and TEL + cell lines treated with DMSO/PARGi. b , Representative IF images of HIRA-YFP localization in U2OS cells after exposure to 30J/m 2 ultra-violet C (UV-C) and 10 Gy ionizing irradiation (γIR). 5μM PARGi was added for 30 mins following irradiation. c , Left: Western blot validation of RPA70 knockdown in U2OS cells. Middle: Representative IF images of HIRA-YFP localization at telomeres in U2OS cells after RPA70 knockdown. Right: Quantification of HIRA-YFP localization to telomeres in indicated conditions from N = 2 independent assays. d , Western blot validation of HIRA, CABIN1 and UBN1 siRNA knockdown in U2OS cells. e , Graphs of CldU/IdU tract distribution of > 30 telomeric fibers in NT siRNA and HIRA siRNA transfected U2OS-TRF1-FokI cells. f , Representative IF images and quantification of BrdU synthesis at telomeres in indicated cell lines that are transfected with WT-TRF1-FokI and HIRA siRNA. All inhibitor treatments, 5 μM/4hrs. All scale bars in IF panels=5μm. Unless otherwise stated, (n) is the number of cells analyzed and the number of independent assays (N) conducted is represented by black circles. Uncropped blots for c-d .

    Journal: Nature structural & molecular biology

    Article Title: Regulation of ALT-associated homology-directed repair by polyADP-ribosylation

    doi: 10.1038/s41594-020-0512-7

    Figure Lengend Snippet: Selectivity of HIRA for localization to telomeres in ALT cancer cells telomeric DSBs is independent of RPA and is necessary for telomere DNA synthesis. a , Representative IF images of HIRA-YFP localization in ALT + and TEL + cell lines treated with DMSO/PARGi. b , Representative IF images of HIRA-YFP localization in U2OS cells after exposure to 30J/m 2 ultra-violet C (UV-C) and 10 Gy ionizing irradiation (γIR). 5μM PARGi was added for 30 mins following irradiation. c , Left: Western blot validation of RPA70 knockdown in U2OS cells. Middle: Representative IF images of HIRA-YFP localization at telomeres in U2OS cells after RPA70 knockdown. Right: Quantification of HIRA-YFP localization to telomeres in indicated conditions from N = 2 independent assays. d , Western blot validation of HIRA, CABIN1 and UBN1 siRNA knockdown in U2OS cells. e , Graphs of CldU/IdU tract distribution of > 30 telomeric fibers in NT siRNA and HIRA siRNA transfected U2OS-TRF1-FokI cells. f , Representative IF images and quantification of BrdU synthesis at telomeres in indicated cell lines that are transfected with WT-TRF1-FokI and HIRA siRNA. All inhibitor treatments, 5 μM/4hrs. All scale bars in IF panels=5μm. Unless otherwise stated, (n) is the number of cells analyzed and the number of independent assays (N) conducted is represented by black circles. Uncropped blots for c-d .

    Article Snippet: Denatured genomic DNA was incubated with 2 μg anti-IgG (Sigma) or anti-BrdU antibody (BD) diluted in immunoprecipitation buffer (0.0625 % (vol/vol) Triton X-100 in PBS) with rotation overnight and at 4 °C.

    Techniques: Recombinase Polymerase Amplification, DNA Synthesis, Irradiation, Western Blot, Transfection

    PARylation is an early and direct mediator of TRF1-FokI DSB formation. a , [ADP/ATP] ratio in DMSO/PARGi treated WT-TRF1-FokI U2OS cells. Cells were treated with 1.5 mM/1 hr MMS. ( b ) Representative IF images and quantification of PAR at WT-TRF1-FokI DSBs after PARGi, PARGi-PARPi or TNKS1 knockdown. c , Representative IF images and quantification showing GFP-PARP1 localization in WT-TRF1-FokI cells treated with PARPi, PARGi or both. d , Representative IF images and quantification showing GFP-PARG localization in WT-TRF1-FokI U2OS cells. e , Left: Representative IF images and quantification of telomere foci size per cell in VA13 and Hela LT cells transfected with WT-TRF1-FokI from N = 2 independent assays. f , Representative stills of telomere (eGFP-TRF1) movement in U2OS cells treated with DMSO, PARPi or PARGi. Graph displays the cumulative Mean Squared Displacement (MSD) of 100 telomeres. g , Top: Schematic of DNA combing in G2-synchronized WT-TRF1-FokI cells treated with DMSO, PARPi, PARGi, or co-treated with PARPi and PARGi. Left: Quantification of telomeric fiber length of combined pulses. Right: Violin plot analysis of fork velocity. h , Graphs of CldU/IdU tract distribution of telomeric fibers in inhibitor treated U2OS-TRF1-FokI cells. n refers to the number of fibers containing TTAGGG signals analyzed from N = 2 independent assays. i , Representative IF images and quantification of BrdU synthesis at telomeres in the indicated cell lines after transfection with WT-TRF1-FokI and treated with PARGi or PARPi. j , Representative IF images and quantification of PCNA and ( k ) POLD3 localization at WT-TRF1-FokI telomeres treated with treated with DMSO, PARPi, PARGi, PARGi −Me or PARPi-PARGi. All inhibitor treatments, 5μM/4hrs unless otherwise indicated. All scale bars in IF panels=5μm. All graphed data in the figure are mean ± s.e.m. Unless otherwise stated, (n) .

    Journal: Nature structural & molecular biology

    Article Title: Regulation of ALT-associated homology-directed repair by polyADP-ribosylation

    doi: 10.1038/s41594-020-0512-7

    Figure Lengend Snippet: PARylation is an early and direct mediator of TRF1-FokI DSB formation. a , [ADP/ATP] ratio in DMSO/PARGi treated WT-TRF1-FokI U2OS cells. Cells were treated with 1.5 mM/1 hr MMS. ( b ) Representative IF images and quantification of PAR at WT-TRF1-FokI DSBs after PARGi, PARGi-PARPi or TNKS1 knockdown. c , Representative IF images and quantification showing GFP-PARP1 localization in WT-TRF1-FokI cells treated with PARPi, PARGi or both. d , Representative IF images and quantification showing GFP-PARG localization in WT-TRF1-FokI U2OS cells. e , Left: Representative IF images and quantification of telomere foci size per cell in VA13 and Hela LT cells transfected with WT-TRF1-FokI from N = 2 independent assays. f , Representative stills of telomere (eGFP-TRF1) movement in U2OS cells treated with DMSO, PARPi or PARGi. Graph displays the cumulative Mean Squared Displacement (MSD) of 100 telomeres. g , Top: Schematic of DNA combing in G2-synchronized WT-TRF1-FokI cells treated with DMSO, PARPi, PARGi, or co-treated with PARPi and PARGi. Left: Quantification of telomeric fiber length of combined pulses. Right: Violin plot analysis of fork velocity. h , Graphs of CldU/IdU tract distribution of telomeric fibers in inhibitor treated U2OS-TRF1-FokI cells. n refers to the number of fibers containing TTAGGG signals analyzed from N = 2 independent assays. i , Representative IF images and quantification of BrdU synthesis at telomeres in the indicated cell lines after transfection with WT-TRF1-FokI and treated with PARGi or PARPi. j , Representative IF images and quantification of PCNA and ( k ) POLD3 localization at WT-TRF1-FokI telomeres treated with treated with DMSO, PARPi, PARGi, PARGi −Me or PARPi-PARGi. All inhibitor treatments, 5μM/4hrs unless otherwise indicated. All scale bars in IF panels=5μm. All graphed data in the figure are mean ± s.e.m. Unless otherwise stated, (n) .

    Article Snippet: Denatured genomic DNA was incubated with 2 μg anti-IgG (Sigma) or anti-BrdU antibody (BD) diluted in immunoprecipitation buffer (0.0625 % (vol/vol) Triton X-100 in PBS) with rotation overnight and at 4 °C.

    Techniques: Transfection

    Selective AKT inhibition by MK-2206 diminishes TIC activity and survival in vitro. a Three subpopulations in SW480 cells based on CD133 expression. Representative FACS blot. b Tumor growth of 50,000 prospectively isolated CD133 subpopulations injected subcutaneously in NSG ( n = 4). c – g SW480 cells were treated with 1, 5, or 10 µM MK-2206 for the indicated times. c Western blot analysis after 72 h of treatment. d Cell proliferation (MTT assay). e Fold change of CD133 high -expressing cells detected via FACS. f Early apoptotic cells (AnnexinV + /7-AAD − ) in CD133 subpopulations determined via FACS. ( g ) Cell cycle phase distribution in CD133 + bulk cells detected via BrdU incorporation. h Tumorsphere formation in CRC cell lines. Data are expressed as mean and standard deviation. * p

    Journal: Annals of Surgical Oncology

    Article Title: Selective AKT Inhibition by MK-2206 Represses Colorectal Cancer-Initiating Stem Cells

    doi: 10.1245/s10434-016-5218-z

    Figure Lengend Snippet: Selective AKT inhibition by MK-2206 diminishes TIC activity and survival in vitro. a Three subpopulations in SW480 cells based on CD133 expression. Representative FACS blot. b Tumor growth of 50,000 prospectively isolated CD133 subpopulations injected subcutaneously in NSG ( n = 4). c – g SW480 cells were treated with 1, 5, or 10 µM MK-2206 for the indicated times. c Western blot analysis after 72 h of treatment. d Cell proliferation (MTT assay). e Fold change of CD133 high -expressing cells detected via FACS. f Early apoptotic cells (AnnexinV + /7-AAD − ) in CD133 subpopulations determined via FACS. ( g ) Cell cycle phase distribution in CD133 + bulk cells detected via BrdU incorporation. h Tumorsphere formation in CRC cell lines. Data are expressed as mean and standard deviation. * p

    Article Snippet: To determine the cell cycle distribution, 48 and 72 h after treatment cells were pulsed with bromodeoxyuridine (BrdU) for 8 h, stained with anti-CD133-PE followed by fixation/permeabilization and anti-BrdU and DNA content (7AAD) staining using an FITC BrdU flow kit (BD, Germany), according to the manufacturer’s instructions.

    Techniques: Inhibition, Activity Assay, In Vitro, Expressing, FACS, Isolation, Injection, Western Blot, MTT Assay, BrdU Incorporation Assay, Standard Deviation

    Synergistic antitumoral effects of 5-fluorouracil and MK-2206 in CRC. a – c SW480, HCT and Cx-1 cells were treated with either 5 µM MK-2206, 10 µM 5-fluorouracil, or in combination. a Cell proliferation was determined after 48 h (MTT assay). b SW480 tumorspheres scored after 14 days of treatment. c Percentage of CD133 high -SW480 cells in G1 phase after 72 h of treatment detected via an 8 h BrdU pulse. Data are expressed as mean and standard deviation. * p

    Journal: Annals of Surgical Oncology

    Article Title: Selective AKT Inhibition by MK-2206 Represses Colorectal Cancer-Initiating Stem Cells

    doi: 10.1245/s10434-016-5218-z

    Figure Lengend Snippet: Synergistic antitumoral effects of 5-fluorouracil and MK-2206 in CRC. a – c SW480, HCT and Cx-1 cells were treated with either 5 µM MK-2206, 10 µM 5-fluorouracil, or in combination. a Cell proliferation was determined after 48 h (MTT assay). b SW480 tumorspheres scored after 14 days of treatment. c Percentage of CD133 high -SW480 cells in G1 phase after 72 h of treatment detected via an 8 h BrdU pulse. Data are expressed as mean and standard deviation. * p

    Article Snippet: To determine the cell cycle distribution, 48 and 72 h after treatment cells were pulsed with bromodeoxyuridine (BrdU) for 8 h, stained with anti-CD133-PE followed by fixation/permeabilization and anti-BrdU and DNA content (7AAD) staining using an FITC BrdU flow kit (BD, Germany), according to the manufacturer’s instructions.

    Techniques: MTT Assay, Standard Deviation

    Akt2 deficiency is insufficient to inhibit the development of endometrial carcinoma in Pten +/– mice. ( a ) Histological sections representing the different grades of endometrial neopplasia in Pten +−/− mice (AH, atypical hyperplasia, CIS, carcinoma in situ ). The source of the tissue sections is indicated. ( b ) Incidence of endometrial neoplasia in Pten +/– , Pten +/– Akt2 –/– and wild-type mice. Total number of mice examined in each group is indicated in parentheses. ( c ) BrdU incorporation in the uteri of Pten +/– , Pten +/– Akt2 –/– and wild-type mice. The numbers of mice in each group are indicated in parentheses. P -values are indicated.

    Journal: Oncogene

    Article Title: The effect Akt2 deletion on tumor development in Pten+/− mice

    doi: 10.1038/onc.2011.243

    Figure Lengend Snippet: Akt2 deficiency is insufficient to inhibit the development of endometrial carcinoma in Pten +/– mice. ( a ) Histological sections representing the different grades of endometrial neopplasia in Pten +−/− mice (AH, atypical hyperplasia, CIS, carcinoma in situ ). The source of the tissue sections is indicated. ( b ) Incidence of endometrial neoplasia in Pten +/– , Pten +/– Akt2 –/– and wild-type mice. Total number of mice examined in each group is indicated in parentheses. ( c ) BrdU incorporation in the uteri of Pten +/– , Pten +/– Akt2 –/– and wild-type mice. The numbers of mice in each group are indicated in parentheses. P -values are indicated.

    Article Snippet: The following primary antibodies were used: rabbit anti-pS473-Akt, (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-PTEN (Biosource, Camarillo, CA, USA), mouse anti-BrdU (Dako, Carpinteria, CA, USA) and rabbit anti-cytokeratin 14 (Novocastra Laboratory, Newcastle upon Tyne, UK).

    Techniques: Mouse Assay, In Situ, BrdU Incorporation Assay

    Effect of Akt2 deficiency on tumor development in the adrenal gland and on the number of polyps in the small intestine of Pten +/– mice. ( a ) The diameter of the adrenal glands in 9-month-old female or 12-month-old male, Pten +/– , Pten +/– Akt2 –/– and wild-type mice. The numbers of mice are indicated in parentheses. ( b ) Quantification of BrdU incorporation in the adrenal medulla of Pten +/– and Pten +/– Akt2 –/– mice. BrdU analysis was carried out as described in Figure 1b . ( c ) The deficiency of Akt2 did not reduce the number of polyps in the small intestine of Pten +/– mice. Quantification of the number of intestinal polyps in 9-month-old female and 12-month-old male mice. The number of polyps±s.d. per mouse is shown. The numbers of mice are indicated in parentheses (F, female; M, male).

    Journal: Oncogene

    Article Title: The effect Akt2 deletion on tumor development in Pten+/− mice

    doi: 10.1038/onc.2011.243

    Figure Lengend Snippet: Effect of Akt2 deficiency on tumor development in the adrenal gland and on the number of polyps in the small intestine of Pten +/– mice. ( a ) The diameter of the adrenal glands in 9-month-old female or 12-month-old male, Pten +/– , Pten +/– Akt2 –/– and wild-type mice. The numbers of mice are indicated in parentheses. ( b ) Quantification of BrdU incorporation in the adrenal medulla of Pten +/– and Pten +/– Akt2 –/– mice. BrdU analysis was carried out as described in Figure 1b . ( c ) The deficiency of Akt2 did not reduce the number of polyps in the small intestine of Pten +/– mice. Quantification of the number of intestinal polyps in 9-month-old female and 12-month-old male mice. The number of polyps±s.d. per mouse is shown. The numbers of mice are indicated in parentheses (F, female; M, male).

    Article Snippet: The following primary antibodies were used: rabbit anti-pS473-Akt, (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-PTEN (Biosource, Camarillo, CA, USA), mouse anti-BrdU (Dako, Carpinteria, CA, USA) and rabbit anti-cytokeratin 14 (Novocastra Laboratory, Newcastle upon Tyne, UK).

    Techniques: Mouse Assay, BrdU Incorporation Assay

    Akt2 deficiency does not impair the development of high-grade PIN in Pten +/– mice. ( a ) Incidence of PIN3 and PIN4 lesions in the three prostate lobes (A, anterior, DL, dorsolateral and V, ventral) of Pten +/– and Pten +/– Akt2 –/– mice. The numbers of mice in each group are indicated. ( b ) BrdU incorporation in the prostate lobes of Pten +/– and Pten +/– Akt2 –/– mice. The numbers of mice in each group are indicated in parentheses. BrdU-positive cells were counted as described in Materials and methods. P -values were calculated for each prostate lobe in each genotype. ( c ) Quantification of K14 staining, from five mice of each genotype as described in Materials and methods.

    Journal: Oncogene

    Article Title: The effect Akt2 deletion on tumor development in Pten+/− mice

    doi: 10.1038/onc.2011.243

    Figure Lengend Snippet: Akt2 deficiency does not impair the development of high-grade PIN in Pten +/– mice. ( a ) Incidence of PIN3 and PIN4 lesions in the three prostate lobes (A, anterior, DL, dorsolateral and V, ventral) of Pten +/– and Pten +/– Akt2 –/– mice. The numbers of mice in each group are indicated. ( b ) BrdU incorporation in the prostate lobes of Pten +/– and Pten +/– Akt2 –/– mice. The numbers of mice in each group are indicated in parentheses. BrdU-positive cells were counted as described in Materials and methods. P -values were calculated for each prostate lobe in each genotype. ( c ) Quantification of K14 staining, from five mice of each genotype as described in Materials and methods.

    Article Snippet: The following primary antibodies were used: rabbit anti-pS473-Akt, (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-PTEN (Biosource, Camarillo, CA, USA), mouse anti-BrdU (Dako, Carpinteria, CA, USA) and rabbit anti-cytokeratin 14 (Novocastra Laboratory, Newcastle upon Tyne, UK).

    Techniques: Mouse Assay, BrdU Incorporation Assay, Staining

    Analysis of chondrocyte proliferation in the limbs by BrdU labeling. ( A – D ) Immunohistochemistry of tibiae from BrdU-labeled wild-type ( A ), Runx2 –/– ( B ), Runx2 –/– 3 +/– ( C ), and Runx2 –/– 3 –/– ( D ) embryos at E18.5 using anti-BrdU antibody. The sections were counterstained with toluidine blue. Magnified views of the boxed regions ( a,b,c ) are shown in the same columns. ( A,B ) In wild-type and Runx2 –/– tibiae, the boxed regions a, b , and c represent resting, proliferating, and hypertrophic chondrocytes, respectively. ( C,D ) In the Runx2 –/– 3 +/– and Runx2 –/– 3 –/– tibiae, the boxed regions a, b , and c represent chondrocytes in the epiphyses, metaphyses, and diaphyses, respectively. The growth plates were well formed in wild-type tibiae ( A ) and in Runx2 –/– tibiae ( B ) but not in Runx2 –/– 3 +/– tibiae ( C ) or Runx2 –/– 3 –/– tibiae ( D ). ( Ab – Db ) The columnar alignment of chondrocytes, which is seen in the layer of proliferating chondrocytes, is well formed in the wild-type and Runx2 –/– tibiae, deformed in the Runx2 –/– 3 +/– tibiae, and completely absent in the Runx2 –/– 3 –/– tibiae. ( C,D ) The diaphyses of Runx2 –/– 3 +/– tibiae were composed of slightly enlarged chondrocytes, whereas the entire tibiae of Runx2 –/– 3 –/– embryos were composed of homogeneously small chondrocytes. ( E – I ) Measurement of the frequency of BrdU-positive cells ( E,F ), cell number ( G ), matrix area ( H ), and cell size ( I ) using the tibiae of six wild-type embryos, two Runx3 –/– embryos, six Runx2 –/– embryos, one Runx2 –/– 3 +/– embryo, and two Runx2 –/– 3 –/– embryos. We measured these parameters in each region shown in A – D . ( A,B ) In wild-type and Runx2 –/– tibiae, the regions I, II, and III represent resting, proliferating, and hypertrophic and terminal hypertrophic chondrocytes, respectively. ( C,D ) In the Runx2 –/– 3 +/– and Runx2 –/– 3 –/– tibiae, the regions I, II, and III were arbitrarily determined in the proximal half of the tibiae and represent chondrocytes in the epiphysial, metaphysical, and diaphysial parts of the tibiae, respectively. ( F ) We also counted the number of BrdU-positive cells in femurs, because chondrocyte maturation in Runx2 –/– femurs is more severely inhibited than that in Runx2 –/– ). ( F ) In the measurement of BrdU-positive cells in whole tibiae and femurs, we counted the number of BrdU-positive cells and the total number of cells in regions I and II in wild-type and Runx2 –/– mice and in regions I, II, and III in Runx2 –/– 3 +/– and Runx2 –/– 3 –/– mice, and the mean of the percentage of BrdU-positive cells ± standard deviation (std. dev.) is shown. We measured all of the parameters in three sections of each bone, and the mean ± std. dev. is shown. (*) p

    Journal: Genes & Development

    Article Title: Runx2 and Runx3 are essential for chondrocyte maturation, and Runx2 regulates limb growth through induction of Indian hedgehog

    doi: 10.1101/gad.1174704

    Figure Lengend Snippet: Analysis of chondrocyte proliferation in the limbs by BrdU labeling. ( A – D ) Immunohistochemistry of tibiae from BrdU-labeled wild-type ( A ), Runx2 –/– ( B ), Runx2 –/– 3 +/– ( C ), and Runx2 –/– 3 –/– ( D ) embryos at E18.5 using anti-BrdU antibody. The sections were counterstained with toluidine blue. Magnified views of the boxed regions ( a,b,c ) are shown in the same columns. ( A,B ) In wild-type and Runx2 –/– tibiae, the boxed regions a, b , and c represent resting, proliferating, and hypertrophic chondrocytes, respectively. ( C,D ) In the Runx2 –/– 3 +/– and Runx2 –/– 3 –/– tibiae, the boxed regions a, b , and c represent chondrocytes in the epiphyses, metaphyses, and diaphyses, respectively. The growth plates were well formed in wild-type tibiae ( A ) and in Runx2 –/– tibiae ( B ) but not in Runx2 –/– 3 +/– tibiae ( C ) or Runx2 –/– 3 –/– tibiae ( D ). ( Ab – Db ) The columnar alignment of chondrocytes, which is seen in the layer of proliferating chondrocytes, is well formed in the wild-type and Runx2 –/– tibiae, deformed in the Runx2 –/– 3 +/– tibiae, and completely absent in the Runx2 –/– 3 –/– tibiae. ( C,D ) The diaphyses of Runx2 –/– 3 +/– tibiae were composed of slightly enlarged chondrocytes, whereas the entire tibiae of Runx2 –/– 3 –/– embryos were composed of homogeneously small chondrocytes. ( E – I ) Measurement of the frequency of BrdU-positive cells ( E,F ), cell number ( G ), matrix area ( H ), and cell size ( I ) using the tibiae of six wild-type embryos, two Runx3 –/– embryos, six Runx2 –/– embryos, one Runx2 –/– 3 +/– embryo, and two Runx2 –/– 3 –/– embryos. We measured these parameters in each region shown in A – D . ( A,B ) In wild-type and Runx2 –/– tibiae, the regions I, II, and III represent resting, proliferating, and hypertrophic and terminal hypertrophic chondrocytes, respectively. ( C,D ) In the Runx2 –/– 3 +/– and Runx2 –/– 3 –/– tibiae, the regions I, II, and III were arbitrarily determined in the proximal half of the tibiae and represent chondrocytes in the epiphysial, metaphysical, and diaphysial parts of the tibiae, respectively. ( F ) We also counted the number of BrdU-positive cells in femurs, because chondrocyte maturation in Runx2 –/– femurs is more severely inhibited than that in Runx2 –/– ). ( F ) In the measurement of BrdU-positive cells in whole tibiae and femurs, we counted the number of BrdU-positive cells and the total number of cells in regions I and II in wild-type and Runx2 –/– mice and in regions I, II, and III in Runx2 –/– 3 +/– and Runx2 –/– 3 –/– mice, and the mean of the percentage of BrdU-positive cells ± standard deviation (std. dev.) is shown. We measured all of the parameters in three sections of each bone, and the mean ± std. dev. is shown. (*) p

    Article Snippet: We processed the embryos for histological analysis and detected BrdU incorporation by immunohistochemistry using anti-BrdU antibody (Dako).

    Techniques: Labeling, Immunohistochemistry, Mouse Assay, Standard Deviation

    Effects of β-catenin knockdown on Ras-ERK pathway activation and VPA-induced differentiation and inhibition of proliferation . NPCs were transfected with 100 nM control siRNA or β-catenin siRNA prior to treatment with 1 mM VPA for 48 h. (A) Whole-cell lysates were subjected to immunoblotting to detect presence of β-catenin, Pan-Ras, p-ERK, p21 Cip/WAF1 , Tuj1, EGFR, or β-actin. (B-C) Immunofluorescent labeling of Tuj1or BrdU. Nuclei were counterstained with DAPI.

    Journal: BMC Cell Biology

    Article Title: Valproic acid induces differentiation and inhibition of proliferation in neural progenitor cells via the beta-catenin-Ras-ERK-p21Cip/WAF1 pathway

    doi: 10.1186/1471-2121-9-66

    Figure Lengend Snippet: Effects of β-catenin knockdown on Ras-ERK pathway activation and VPA-induced differentiation and inhibition of proliferation . NPCs were transfected with 100 nM control siRNA or β-catenin siRNA prior to treatment with 1 mM VPA for 48 h. (A) Whole-cell lysates were subjected to immunoblotting to detect presence of β-catenin, Pan-Ras, p-ERK, p21 Cip/WAF1 , Tuj1, EGFR, or β-actin. (B-C) Immunofluorescent labeling of Tuj1or BrdU. Nuclei were counterstained with DAPI.

    Article Snippet: The cells were incubated in blocking solution (10% normal goat serum in PBS) for 30 min followed by incubation with anti-Tuj1, anti-BrdU (Dako Co., Carpinteria, CA), anti-Flag (Sigma-Aldrich), or anti-p21Cip/WAF1 in blocking solution at 4°C overnight.

    Techniques: Activation Assay, Inhibition, Transfection, Labeling

    Effect of VPA on activities of ERK pathway components and on expression of p21 Cip/WAF1 and Tuj1 . NPCs grown in 10 ng/ml bFGF were treated with 1 mM VPA for 48 h. (A) Whole-cell lysates were subjected to immunoblotting for analysis of the presence of p-ERK, p-MEK, p-Raf-1, p21 Cip/WAF1 , Tuj1, or β-actin. Alternatively, NPCs were processed for immunofluorescent labeling of (B) p21 Cip/WAF1 (green), (C) BrdU (red), or (D) Tuj1 (green). Nuclei were counterstained with DAPI (blue). All images are 200× magnifications. The percentage of cells that exhibited nuclear p21 Cip/WAF1 in the presence and absence of VPA is presented, along with the percentage of BrdU-positive and Tuj1-positive cells. Error bars indicate the standard deviations of three independent experiments. Data represent mean ± SD of three separate experiments. P***

    Journal: BMC Cell Biology

    Article Title: Valproic acid induces differentiation and inhibition of proliferation in neural progenitor cells via the beta-catenin-Ras-ERK-p21Cip/WAF1 pathway

    doi: 10.1186/1471-2121-9-66

    Figure Lengend Snippet: Effect of VPA on activities of ERK pathway components and on expression of p21 Cip/WAF1 and Tuj1 . NPCs grown in 10 ng/ml bFGF were treated with 1 mM VPA for 48 h. (A) Whole-cell lysates were subjected to immunoblotting for analysis of the presence of p-ERK, p-MEK, p-Raf-1, p21 Cip/WAF1 , Tuj1, or β-actin. Alternatively, NPCs were processed for immunofluorescent labeling of (B) p21 Cip/WAF1 (green), (C) BrdU (red), or (D) Tuj1 (green). Nuclei were counterstained with DAPI (blue). All images are 200× magnifications. The percentage of cells that exhibited nuclear p21 Cip/WAF1 in the presence and absence of VPA is presented, along with the percentage of BrdU-positive and Tuj1-positive cells. Error bars indicate the standard deviations of three independent experiments. Data represent mean ± SD of three separate experiments. P***

    Article Snippet: The cells were incubated in blocking solution (10% normal goat serum in PBS) for 30 min followed by incubation with anti-Tuj1, anti-BrdU (Dako Co., Carpinteria, CA), anti-Flag (Sigma-Aldrich), or anti-p21Cip/WAF1 in blocking solution at 4°C overnight.

    Techniques: Expressing, Labeling

    Effects of bFGF and p21 Cip/WAF1 siRNA on regulation of the VPA-induced differentiation and inhibition of proliferation in NPCs (A-B) . NPCs were treated with 1 mM VPA for 48 h in the presence or absence of 10 ng/ml bFGF. (C-D) NPCs were transfected with 100 nM p21 Cip/WAF1 siRNA prior to treatment with 1 mM VPA for 48 h in the presence of 10 ng/ml bFGF. Whole-cell lysates were subjected to immunoblotting to detect p21 Cip/WAF1 , Tuj1, or β-actin. Alternatively, cells were processed for immunofluorescent labeling to detect the presence of Tuj1 (green), p21 Cip/WAF1 (green), or BrdU (red). Nuclei were counterstained with DAPI. Images are 400× magnifications. (E) NPCs were treated with 1 mM VPA for 48 h in the presence of 10 ng/ml bFGF. Cells were processed for immunofluorescent labeling to detect the presence of p21 Cip/WAF1 (green) or BrdU (red). Nuclei were counterstained with DAPI. Magnification is 1200×.

    Journal: BMC Cell Biology

    Article Title: Valproic acid induces differentiation and inhibition of proliferation in neural progenitor cells via the beta-catenin-Ras-ERK-p21Cip/WAF1 pathway

    doi: 10.1186/1471-2121-9-66

    Figure Lengend Snippet: Effects of bFGF and p21 Cip/WAF1 siRNA on regulation of the VPA-induced differentiation and inhibition of proliferation in NPCs (A-B) . NPCs were treated with 1 mM VPA for 48 h in the presence or absence of 10 ng/ml bFGF. (C-D) NPCs were transfected with 100 nM p21 Cip/WAF1 siRNA prior to treatment with 1 mM VPA for 48 h in the presence of 10 ng/ml bFGF. Whole-cell lysates were subjected to immunoblotting to detect p21 Cip/WAF1 , Tuj1, or β-actin. Alternatively, cells were processed for immunofluorescent labeling to detect the presence of Tuj1 (green), p21 Cip/WAF1 (green), or BrdU (red). Nuclei were counterstained with DAPI. Images are 400× magnifications. (E) NPCs were treated with 1 mM VPA for 48 h in the presence of 10 ng/ml bFGF. Cells were processed for immunofluorescent labeling to detect the presence of p21 Cip/WAF1 (green) or BrdU (red). Nuclei were counterstained with DAPI. Magnification is 1200×.

    Article Snippet: The cells were incubated in blocking solution (10% normal goat serum in PBS) for 30 min followed by incubation with anti-Tuj1, anti-BrdU (Dako Co., Carpinteria, CA), anti-Flag (Sigma-Aldrich), or anti-p21Cip/WAF1 in blocking solution at 4°C overnight.

    Techniques: Inhibition, Transfection, Labeling

    Effects of VPA or β-catenin on EGFR, β-catenin, and Ras regulation . (A-B) NPCs grown in 10 ng/ml bFGF were treated with 1 mM VPA for 48 h or different periods of time. (C-D) NPCs were transfected with pcDNA3.0 or Flag-β-catenin-pcDNA3.0 and grown in the presence of 10 ng/ml bFGF. (A-C) Whole-cell lysates were then subjected to immunoblotting for detection of p-GSK3β (Ser-9), β-catenin, Pan-Ras, p-ERK, p21 Cip/WAF1 , Tuj1, EGFR, or β-actin. (D) Immunofluorescent labeling was performed for Flag or BrdU. Nuclei were counterstained with DAPI.

    Journal: BMC Cell Biology

    Article Title: Valproic acid induces differentiation and inhibition of proliferation in neural progenitor cells via the beta-catenin-Ras-ERK-p21Cip/WAF1 pathway

    doi: 10.1186/1471-2121-9-66

    Figure Lengend Snippet: Effects of VPA or β-catenin on EGFR, β-catenin, and Ras regulation . (A-B) NPCs grown in 10 ng/ml bFGF were treated with 1 mM VPA for 48 h or different periods of time. (C-D) NPCs were transfected with pcDNA3.0 or Flag-β-catenin-pcDNA3.0 and grown in the presence of 10 ng/ml bFGF. (A-C) Whole-cell lysates were then subjected to immunoblotting for detection of p-GSK3β (Ser-9), β-catenin, Pan-Ras, p-ERK, p21 Cip/WAF1 , Tuj1, EGFR, or β-actin. (D) Immunofluorescent labeling was performed for Flag or BrdU. Nuclei were counterstained with DAPI.

    Article Snippet: The cells were incubated in blocking solution (10% normal goat serum in PBS) for 30 min followed by incubation with anti-Tuj1, anti-BrdU (Dako Co., Carpinteria, CA), anti-Flag (Sigma-Aldrich), or anti-p21Cip/WAF1 in blocking solution at 4°C overnight.

    Techniques: Transfection, Labeling

    Effects of VPA on differentiation and proliferation in cerebral cortex of the developing embryo . (A) Coronal sections of E15.5 rat embryo cerebral cortex immunofluorescently labelled for Tuj1 (green) and PCNA (red). (B) Rats at E13.5 of gestation were intravenously injected with 200 mg/kg VPA or PBS at 0 and 24 h and sacrificed. Immunofluorescence labeling of Tuj1 (green) and PCNA (red) was performed on coronal sections of embryonic brains of the E15.5 rat. The three layers of the cerebral cortex – the cortical plate (CP), the intermediate zone (IMZ), and the subventricular zone (SVZ) above the lateral ventricle (LV)- can be distinguished. The white arrow indicates the direction of migration of the differentiating cells (bar = 25 μm). (C) NPCs from E14 embryos were incubated in the presence of 10 ng/ml bFGF in the presence or absence of 1 mM VPA for 48 h. Immunofluorescence labeling was performed with anti-Tuj1 and anti-BrdU. Nuclei were counterstained with DAPI.

    Journal: BMC Cell Biology

    Article Title: Valproic acid induces differentiation and inhibition of proliferation in neural progenitor cells via the beta-catenin-Ras-ERK-p21Cip/WAF1 pathway

    doi: 10.1186/1471-2121-9-66

    Figure Lengend Snippet: Effects of VPA on differentiation and proliferation in cerebral cortex of the developing embryo . (A) Coronal sections of E15.5 rat embryo cerebral cortex immunofluorescently labelled for Tuj1 (green) and PCNA (red). (B) Rats at E13.5 of gestation were intravenously injected with 200 mg/kg VPA or PBS at 0 and 24 h and sacrificed. Immunofluorescence labeling of Tuj1 (green) and PCNA (red) was performed on coronal sections of embryonic brains of the E15.5 rat. The three layers of the cerebral cortex – the cortical plate (CP), the intermediate zone (IMZ), and the subventricular zone (SVZ) above the lateral ventricle (LV)- can be distinguished. The white arrow indicates the direction of migration of the differentiating cells (bar = 25 μm). (C) NPCs from E14 embryos were incubated in the presence of 10 ng/ml bFGF in the presence or absence of 1 mM VPA for 48 h. Immunofluorescence labeling was performed with anti-Tuj1 and anti-BrdU. Nuclei were counterstained with DAPI.

    Article Snippet: The cells were incubated in blocking solution (10% normal goat serum in PBS) for 30 min followed by incubation with anti-Tuj1, anti-BrdU (Dako Co., Carpinteria, CA), anti-Flag (Sigma-Aldrich), or anti-p21Cip/WAF1 in blocking solution at 4°C overnight.

    Techniques: Injection, Immunofluorescence, Labeling, Migration, Incubation