rabbit anti-ki67 Search Results


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  • 99
    Thermo Fisher rabbit anti ki67
    Cyclin E expression increases pituitary proliferation index. A, Cohorts of Tg-PCE mice and their control sibs (Wt) were investigated at 2 yr of age for a pituitary phenotype. Six transgenic pituitaries of a total of 17 were found to have abnormal pituitaries, three with IL hyperplasia (C) and three with AL adenomas (D–F). B, Quantitation of cyclin E and <t>Ki67-positive</t> cells in adult ILs of Tg-PCE transgenic pituitaries compared with their Wt control sibs. Cyclin E-positive and Ki67-positive (a marker of proliferation) cells were identified by immunohistochemistry on pituitary sections of 8-month-old and 2-yr-old mice as indicated. For each group, the number of positive cells was counted on duplicate sections from eight to 10 different mice. F, Summary of marker expression in the three AL adenomas. Only one tumor was positive for the differentiation markers Pit-1, PRL, and GH.
    Rabbit Anti Ki67, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 849 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti ki67
    TLX is essential for Oct-3/4 expression in hypoxia. A , immunoblot analysis for TLX and Oct-3/4 in AHP cells cultured in the proliferating ( FGF +) or differentiating conditions ( FGF −) upon different time intervals of hypoxia. GAPDH was used as loading control. B , immunofluorescence analysis for TLX ( green ) and Oct-3/4 ( red ) in AHP cells cultured in the presence or absence of FGF under normoxia or 24 h hypoxia. C , comparison of TLX, Oct-3/4, GFAP, and Map2a protein levels in AHP cells cultured in proliferating conditions and depleted of TLX by shRNA ( Sh TLX ), or differentiating conditions, overexpressing TLX. GAPDH was used as loading control. A control shRNA ( Sh Ctrl ) and the vector control ( Vec ctrl ) were used as standards. D , immunoblot analysis for comparison of proliferation markers <t>Ki67,</t> pAkt, and total Akt, along with TLX, Oct-3/4, and GFAP differentiation marker in cells cultured under differentiating condition upon transfection of TLX singly or with Oct-3/4 shRNA ( Oct4 sh ) and TLX shRNA ( TLX sh ) alone or in combination with Oct-3/4 shRNA. GAPDH was used as loading control. E , immunoblot analysis for comparison of proliferation markers Ki67, pAkt, and total Akt, along with TLX, Oct-3/4 in cells cultured under differentiating conditions upon transfection of TLX, Oct-3/4 alone or in combination with TLX shRNA. GAPDH was used as loading control. F, table depicting the percentage of positive cells for BrdU staining in normoxia upon TLX overexpression ( TLX ) with or without Oct-3/4 depletion ( TLX + Oct Sh ) and hypoxia upon TLX depletion alone ( TLX sh ) or in combination with Oct-3/4 knockdown ( TLX sh + Oct Sh ).
    Anti Ki67, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 371 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam rabbit anti ki67
    Cell proliferation in the hippocampal SGZ is upregulated by acute restraint stress. (A) To compare the extent of cell proliferation, sectioned brain samples were immunostained with <t>Ki67</t> (green) and mature neuronal marker, NeuN, antibodies (red). (B) Quantification of Ki67-positive cells in acute RST stress mice and the control group. Although extended subjection to restraint stress did not significantly change the number of proliferating cells, RST stress upregulated the number of Ki67-positive cells in the SGZ. * P
    Rabbit Anti Ki67, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 3552 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc antibodies ki67
    Combination Epigenetic Treatment Reduces Lung Tumor Burden and Progression in Mouse Models of NSCLC (A) Representative H E-stained images of lung sections from mice treated with mock or Aza + ITF-2357. Scale bar, 100 μm. (B) Quantitation of total tumor area occupied by lesions in lungs of LSL-Kras G12D mice treated with mock or Aza + ITF-2357. Data are presented as mean ± SEM (p value determined by two-tailed t test; n = 6 mock and n = 7 Aza + ITF-2357 mice per group/2 sections analyzed per mouse). (C) Representative <t>Ki67-stained</t> IHC images of lung sections from mice treated with mock or Aza + ITF-2357 (n = 5 per group). Scale bar, 100 μm. (D) Lewis Lung Carcinoma (LLC) tumor weights of subcutaneous explants from 1-month mock- and Aza + ITF-2357-treated mice (n = 19 mice per group; error bars, SEM). (E) Representative H E-stained images of lung metastasis from LLC mice obtained from 1-month mock- and Aza + ITF-2357-treated mice (n = 12 mice per group), with the indicated percentage frequency of metastasis. (F) Volcano plot of relative RNA expression from LSL-Kras G12D mouse lung tumors treated for 3 months with Aza + ITF-2357 as compared to mock mice. Genes in upper left and right quadrants are significantly differentially expressed (microarray, n = 2 per group). (G) GSEA (KEGG, REACTOME, and HALLMARK) pathway distribution for Aza + ITF-2357 versus mock tumors from LSL-Kras G12D mice. Horizontal line denotes FDR significance cutoff of 0.25. Immune- and cell cycle-related gene sets are demarcated by green and red dots, respectively (microarray, n = 2 per group). (H) Gene sets upregulated in LSL-Kras G12D mice (FDR
    Antibodies Ki67, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Novocastra rabbit anti ki67
    In vivo potential tumorigenesis assay design, as well as percent survival and <t>Ki67</t> expression of late-passage hNPCs and their neuronal progeny. ( A ) Experimental timeline, wherein 3 month old NOD- scid mice were grafted with either hNPCs or MACS-purified neurons (N) in the striatum, and allowed to survive for 6 months. ( B,C ) Representative images of GFP+ surviving cells in mice 6 months after grafting with either hNPCs (scale bar = 10 μm) ( B ) or immature neurons (scale bar = 10 μm) ( C ). ( D ) Difference in overall donor cell survival between cell groups is not significant. ( E ) Migration of implanted cells was estimated on the basis of “migratory profiles characterized by fusiform-shaped cell bodies with single leading and/or trailing processes” (Zheng et al. 38 ). For both cell groups, the total number of GFP+ cells displaying a migratory morphology was counted per the total number of GFP+ cells. Both cell groups displayed migratory characteristics. ( F ) Proliferative status of both cell groups is very low, as indicated by Ki67 immuno-reactivity, particularly in the neuron group. (Ctx = cortex, Str = striatum) Means + s.e.m. P > 0.05, unpaired two-tailed t test.
    Rabbit Anti Ki67, supplied by Novocastra, used in various techniques. Bioz Stars score: 93/100, based on 708 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam rabbit anti ki67 antibodies
    CFC neural cultures show progenitor pool depletion, early maturation, and imbalance of neural cell types (A) Western blot analysis of the components of the Ras/MAPK pathway and its phosphorylation status, and quantification of the blots (B) from protein lysates extracted from CFC and control NPC cultures. The results were normalized with their corresponding α-tubulin values (assigned a value of 1) and are mean ±SD of two independent experiments (C) Double immunostaining for BrdU (green) and <t>Ki67</t> (red) of early neuronal progenitors within 48h of differentiation; progenitors that exited the cell cycle, %(BrdU + Ki67 − )/BrDU + cells (orange arrows) were quantified. (D) Cleaved caspase 3 staining for cell death of the same NPCs and subsequent quantification. (E) Immunostaining of TUJ1, MAP2, NEUN, GABA, and TBR1 of CFC lines and control lines at week 1 of neural differentiation. Quantification of these stainings are shown as percentage of TUJ1 + cells (F), MAP2 + cells (G), NEUN + cells (H), average neurite length of TUJ1+ cells (I), average number of neurites per TUJ1 + cell (J), percentage of GABA + cells (K), and TBR1+ cells (L). (M) mRNA levels of MAP2 , deep layer markers TBR1 , CTIP2, and FEZF2 , upper layer markers CUX1 and SATB2 , and GABA precursors GAD1 and GAD2 in one-week old neural cultures. ΔCt values were calculated by normalizing the average Ct value of each cell line by the average Ct value of GUSB of the same cell line; ΔΔCt was calculated by normalizing the ΔCt value of each line to the average ΔCt value of the control lines. (N) Immunostaining of TUJ1, GFAP, CD44, S100, MAP2, FOXP2, and CUX1 at week 5 of neural differentiation. Quantification of these stainings is shown as a percentage of cells expressing GFAP (O), MAP2 (P), FOXP2 (Q) and CUX1 (R). (S) mRNA level analysis of astroglial cell markers GFAP , S100B , CD44 , neuronal marker MAP2 , deep layer markers TBR1 , CTIP2, and FEZF2 , upper layer markers CUX1 and SATB2 in five-week old neural cultures. ΔCt values were calculated by normalizing the average Ct value of each cell lines by the average Ct value of GUSB of the same cell line; ΔΔCt was calculated by normalizing the ΔCt value of each line to the average ΔCt value of the control lines. For all immunostainings depicted, at least three fields per coverslip from three independent experiments were counted. For all scatter plots illustrated, values represent mean±SD per condition, calculated with repeated measures ANOVA, accounting for biological and technical replicate values. Number of subclones and independent experiments performed are detailed in Supplementary Table 3 . Values for mean and standard deviation of each control and CFC subject for each experiment are detailed in Supplementary Table 7 . p ≤0.05 (*), p ≤0.01 (**), p ≤0.001 (***).
    Rabbit Anti Ki67 Antibodies, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Abcam rabbit anti ki 67
    Immunohistochemistry for <t>Ki-67</t> in the dentate gyrus of the vehicle (A), scopolamine (SCO) (B), SCO+ vanillin (C), and SCO+4-hydroxybenzyl alcohol (4-HBA) (D) groups. In the vehicle group, Ki-67–immunoreactive cells (arrows) are shown in the subgranular zone. Ki-67-immunoreactive cells are significantly decreased in the SCO group; however, they are increased in the SCO+vanillin and SCO+4-HBA groups. GCL, granule cell layer; ML, molecular layer; PL, polymorphic layer. Scale bar=100 µm. (E) The mean number of Ki-67–immunoreactive cells per group (n=14 per group; * P
    Rabbit Anti Ki 67, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 742 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti ki67 antibody
    Loss of RNF220 inhibited NSC proliferation and promoted differentiation of NSCs. ( A – F ) Loss of RNF220 inhibited NSCs proliferation by EDU incorporation assay ( A – C ) and <t>Ki67</t> immunostaining ( D – F ); the nuclei were marked by DAPI. Scale bar: 50 µm. ( C , F ) The quantification data for ( A , B , D , E ) respectively. ( G – L ) Immunofluorescence analyses of the differentiated neurons from WT and ZC4H2 −/− NSCs for β-tubulin III (TUJ1) ( G – I ) and MAP2 ( J – L ); the nuclei were marked by DAPI. Scale bar: 50 µm. ( I , L ) The quantification data for ( G , H , J , K ), respectively. ( M , N ) Expression of stemness marker genes (Nestin and Vimentin) in WT and RNF220 −/− NSCs. ( O , P ) Expression of neuronal marker genes (TUJ1 and MAP2) in differentiated WT and RNF220 −/− NSCs. β-actin was used as an internal control. ** p
    Anti Ki67 Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 2104 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam rabbit polyclonal anti ki67
    Loss of RNF220 inhibited NSC proliferation and promoted differentiation of NSCs. ( A – F ) Loss of RNF220 inhibited NSCs proliferation by EDU incorporation assay ( A – C ) and <t>Ki67</t> immunostaining ( D – F ); the nuclei were marked by DAPI. Scale bar: 50 µm. ( C , F ) The quantification data for ( A , B , D , E ) respectively. ( G – L ) Immunofluorescence analyses of the differentiated neurons from WT and ZC4H2 −/− NSCs for β-tubulin III (TUJ1) ( G – I ) and MAP2 ( J – L ); the nuclei were marked by DAPI. Scale bar: 50 µm. ( I , L ) The quantification data for ( G , H , J , K ), respectively. ( M , N ) Expression of stemness marker genes (Nestin and Vimentin) in WT and RNF220 −/− NSCs. ( O , P ) Expression of neuronal marker genes (TUJ1 and MAP2) in differentiated WT and RNF220 −/− NSCs. β-actin was used as an internal control. ** p
    Rabbit Polyclonal Anti Ki67, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 374 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher ki 67 rabbit monoclonal antibody supernatant
    Some supporting cells in Ad.MT58A-infected utricles survive for weeks in culture after reentering the cell cycle. ( A ) Diagram depicting the BrdU labeling paradigm. BrdU was either included in the culture medium for the entire culture period after infection with adenovirus, or it was washed out at 5 days post-virus (DPV) and utricles were cultured for an additional 5 d in its absence. ( B–C ) Confocal images show Ad.MT58A-infected utricles (1×10 9 TU/mL) fixed at 10 DPV after being cultured with BrdU from 1–10 DPV (B) or 1–5 DPV (C). Scale bar for B–C, 100 µm. ( D ) Graph shows quantification of the number of BrdU-labeled cells in the sensory epithelium from utricles cultured as depicted in A (gray and white bars). Quantification of 5 DPV BrdU labeling (same as in Fig. 5G) is shown to visualize the decline in BrdU-labeled cells from 5 DPV (black bars) to 10 DPV. Data shown is from co-infection experiments (OKSM), infection with 2×10 8 TU/mL Ad.MT58A (1×M), and infection with 1×10 9 TU/mL Ad.MT58A (5×M). ( E ) Confocal image of an Ad.MT58A-infected utricle (1×10 9 TU/mL) fixed at 21 DPV after being cultured with BrdU from 1–5 DPV. Antibody labeling for BrdU and <t>Ki-67</t> is shown in green and red, respectively. Scale bar, 100 µm. ( F ) Confocal image of an Ad.MT58A-infected utricle (1×10 9 TU/mL) fixed at 21 DPV after being cultured with BrdU from 18–21 DPV. Antibody labeling for BrdU and myosin VIIA is shown in green and magenta, respectively. Scale bar, 100 µm. ( G ) Confocal image of an Ad.MT58A-infected utricle fixed at 10 DPV after switching from growth medium to differentiation medium at 5 DPV. BrdU (green) was included in the medium throughout. Scale bar, 100 µm. ( H ) Graph shows the mean number of BrdU-positive nuclei per sensory epithelium at 5, 10, and 14 DPV for the experiment described in G. White dashed lines demarcate the borders of the sensory epithelium in all panels.
    Ki 67 Rabbit Monoclonal Antibody Supernatant, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 783 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti ki67 antibody epr3610
    Some supporting cells in Ad.MT58A-infected utricles survive for weeks in culture after reentering the cell cycle. ( A ) Diagram depicting the BrdU labeling paradigm. BrdU was either included in the culture medium for the entire culture period after infection with adenovirus, or it was washed out at 5 days post-virus (DPV) and utricles were cultured for an additional 5 d in its absence. ( B–C ) Confocal images show Ad.MT58A-infected utricles (1×10 9 TU/mL) fixed at 10 DPV after being cultured with BrdU from 1–10 DPV (B) or 1–5 DPV (C). Scale bar for B–C, 100 µm. ( D ) Graph shows quantification of the number of BrdU-labeled cells in the sensory epithelium from utricles cultured as depicted in A (gray and white bars). Quantification of 5 DPV BrdU labeling (same as in Fig. 5G) is shown to visualize the decline in BrdU-labeled cells from 5 DPV (black bars) to 10 DPV. Data shown is from co-infection experiments (OKSM), infection with 2×10 8 TU/mL Ad.MT58A (1×M), and infection with 1×10 9 TU/mL Ad.MT58A (5×M). ( E ) Confocal image of an Ad.MT58A-infected utricle (1×10 9 TU/mL) fixed at 21 DPV after being cultured with BrdU from 1–5 DPV. Antibody labeling for BrdU and <t>Ki-67</t> is shown in green and red, respectively. Scale bar, 100 µm. ( F ) Confocal image of an Ad.MT58A-infected utricle (1×10 9 TU/mL) fixed at 21 DPV after being cultured with BrdU from 18–21 DPV. Antibody labeling for BrdU and myosin VIIA is shown in green and magenta, respectively. Scale bar, 100 µm. ( G ) Confocal image of an Ad.MT58A-infected utricle fixed at 10 DPV after switching from growth medium to differentiation medium at 5 DPV. BrdU (green) was included in the medium throughout. Scale bar, 100 µm. ( H ) Graph shows the mean number of BrdU-positive nuclei per sensory epithelium at 5, 10, and 14 DPV for the experiment described in G. White dashed lines demarcate the borders of the sensory epithelium in all panels.
    Anti Ki67 Antibody Epr3610, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 95 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher rabbit anti ki 67
    During normal development, the PDGFR-α-expressing oligodendrocyte progenitor population declines as the number of MBP-expressing mature oligodendrocytes increases. Cortical sections taken from wild-type animals at P0–P60 were stained for the oligodendrocyte progenitor marker PDGFR-α ( a–f ) and the mature oligodendrocyte marker MBP ( g–l ) using immunohistochemistry. Higher-magnification images of sections taken over the period from P0 to P60 stained with PDGFR-α, MBP and the proliferation marker <t>Ki-67</t> demonstrate that proliferating oligodendrocyte progenitors diminish over time as myelination occurs, though proliferative PDGFR-α-expressing progenitors persist at least through 2 months of age ( o–t , arrows). Scale bars = 500 μm ( a, g ) and 50 μm ( a , inset, o ). m, n Western blot analysis of cortical samples taken from animals ranging in age from P0 to P60 was used to characterize PDGFR-α (180 kDa; m ) and MBP (14 kDa; n ) expression.
    Rabbit Anti Ki 67, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 250 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher rabbit monoclonal anti ki67
    Nicotine reduces tubular epithelial cell apoptosis and subsequent proliferation. Immunostaining of cleaved caspase-3 (A, B) and <t>Ki67</t> (D, E) were performed one day and 3 days after I/R, respectively. Panel A represents TEC apoptosis in saline-treated animals (magnification ×40) and panel B represents TEC apoptosis in nicotine pre-treated animals (magnification ×40). Graph C compares the quantification of apoptotic TECs in saline-treated (black bars) or nicotine-treated animals (white bars), either after sham operation (n = 6 in each group) or renal I/R (n = 7 in saline-treated group and n = 10 in nicotine pretreated group). Panels D and E compare Ki-67+ cells between saline-treated animals and nicotine-treated animals (magnification ×40). Graph F compares the quantification of proliferation 3 days after reperfusion (n = 5 in each group). Data are expressed as mean ± SEM, (*) p≤0.05.
    Rabbit Monoclonal Anti Ki67, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 175 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Abcam rabbit anti mouse ki67
    Epithelial proliferation after Nippostrongylus brasiliensis ( N.b. ) infection. ( a ) Representative photographs of lung tissues from naive or infected WT C57BL/6 mice at various time-points following subcutaneous infection of 650 L 3 N.b. . Arrows point to focal areas of hemorrhagic injury. All images were taken at 20× with Dino-Lite Pro Digital Microscope via Dino Capture 2.0 software. ( b ) Quantifications of Ym1 + , Ym1 + Ki-67 + , and Ki-67 + during N.b. infection. For each time-point, 5 randomly selected fields under 20× were counted. ( c ) Kinetic analysis of Ym1 (Cy2) and Ki-67 (Cy3) immunostaining within FFPE distal lung tissues of naïve or N.b. -infected mice at the time-points indicated. 200× magnification. ( d, e ) Images of ( d ) d3 and ( e ) d7 post-infection lung tissues at 400×. Yellow arrowheads indicate Ki-67 + cells and red arrowheads indicate <t>Ki67</t> + /Ym1 + co-staining, respectively. DAPI counterstain is shown in blue. ( f ) Pseudo-color flow plots showing distal lung total live CD45 neg cells gated for EpCAM + cells that were BrdU + at the indicated time-points following N.b. infection. ( g ) Quantification of cells gated as in f isolated from WT C57BL/6 mice. ( h ) Comparison of cells gated as in f from WT, IL-4Rα −/− and RAGγc −/− strains at indicated time points. N=4 mice/group. Mean ±SEM are shown. *p
    Rabbit Anti Mouse Ki67, supplied by Abcam, used in various techniques. Bioz Stars score: 89/100, based on 158 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam rabbit anti human ki67
    Immunohistochemical detection of <t>Ki67</t> in Winnie distal colonic mucosa. A: Immunostaining of Ki67 in the distal colon of untreated C57BL6 mice. Image representative of Ki67 localisation in the distal colon of the four C57BL6 mice examined; B: Distal colonic Ki67 localisation representative of six C57BL6 mice exposed to three cycles of 1% dextran sulphate sodium (DSS); C: Distal colon of Winnie mouse without exposure to three cycles of DSS. Ki67-labelling in the epithelium is visible apically approximately half the crypt length; D: Ki67 immunolabelling of the Winnie distal colon exposed to three cycles of DSS. Crypt base proliferative zone extends approximately two-thirds of the crypt length. Submucosal gland (arrowhead) displays few positive nuclei. Scale bar represents a distance of 50 μm.
    Rabbit Anti Human Ki67, supplied by Abcam, used in various techniques. Bioz Stars score: 89/100, based on 102 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc ki67
    BEZ235 and LY3203414 decrease cellular proliferation and increase differentiation in Apc and Pik3ca mutant colon cancer cells. AP spheroids grown in Matrigel on glass coverslips were treated with NVP-BYL719, BEZ235, LY3203414 or control for 24 hours. A decrease in the percent of cells staining for <t>Ki67</t> was observed in those spheres treated with BEZ235 (p = 0.037) or LY3203414 (p = 0.01), but not NVP-BYL719 (p = 0.74; A). No significant differences were observed in staining for cleaved caspase 3 (B). An increase in staining for keratin 20, a marker of cellular differentiation was observed with BEZ235 and LY3023414 compared to NVP-BEZ719 and control (C).
    Ki67, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1506 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Cyclin E expression increases pituitary proliferation index. A, Cohorts of Tg-PCE mice and their control sibs (Wt) were investigated at 2 yr of age for a pituitary phenotype. Six transgenic pituitaries of a total of 17 were found to have abnormal pituitaries, three with IL hyperplasia (C) and three with AL adenomas (D–F). B, Quantitation of cyclin E and Ki67-positive cells in adult ILs of Tg-PCE transgenic pituitaries compared with their Wt control sibs. Cyclin E-positive and Ki67-positive (a marker of proliferation) cells were identified by immunohistochemistry on pituitary sections of 8-month-old and 2-yr-old mice as indicated. For each group, the number of positive cells was counted on duplicate sections from eight to 10 different mice. F, Summary of marker expression in the three AL adenomas. Only one tumor was positive for the differentiation markers Pit-1, PRL, and GH.

    Journal: Molecular Endocrinology

    Article Title: Cooperation between Cyclin E and p27Kip1 in Pituitary Tumorigenesis

    doi: 10.1210/me.2010-0091

    Figure Lengend Snippet: Cyclin E expression increases pituitary proliferation index. A, Cohorts of Tg-PCE mice and their control sibs (Wt) were investigated at 2 yr of age for a pituitary phenotype. Six transgenic pituitaries of a total of 17 were found to have abnormal pituitaries, three with IL hyperplasia (C) and three with AL adenomas (D–F). B, Quantitation of cyclin E and Ki67-positive cells in adult ILs of Tg-PCE transgenic pituitaries compared with their Wt control sibs. Cyclin E-positive and Ki67-positive (a marker of proliferation) cells were identified by immunohistochemistry on pituitary sections of 8-month-old and 2-yr-old mice as indicated. For each group, the number of positive cells was counted on duplicate sections from eight to 10 different mice. F, Summary of marker expression in the three AL adenomas. Only one tumor was positive for the differentiation markers Pit-1, PRL, and GH.

    Article Snippet: Primary antibodies diluted in 10% normal goat serum-0.2% Tween 20 in PBS were incubated overnight and used at the following dilutions: rabbit-anti-Brg1 1:10 (H-88, Santa Cruz Biotechnology), rabbit-anticyclin E 1:100 (M-20, Santa Cruz Biotechnology), rabbit-anti-Ki67 1:100 (Labvision, Fremont, CA), mouse-anti-p27Kip1.

    Techniques: Expressing, Mouse Assay, Transgenic Assay, Quantitation Assay, Marker, Immunohistochemistry

    TLX is essential for Oct-3/4 expression in hypoxia. A , immunoblot analysis for TLX and Oct-3/4 in AHP cells cultured in the proliferating ( FGF +) or differentiating conditions ( FGF −) upon different time intervals of hypoxia. GAPDH was used as loading control. B , immunofluorescence analysis for TLX ( green ) and Oct-3/4 ( red ) in AHP cells cultured in the presence or absence of FGF under normoxia or 24 h hypoxia. C , comparison of TLX, Oct-3/4, GFAP, and Map2a protein levels in AHP cells cultured in proliferating conditions and depleted of TLX by shRNA ( Sh TLX ), or differentiating conditions, overexpressing TLX. GAPDH was used as loading control. A control shRNA ( Sh Ctrl ) and the vector control ( Vec ctrl ) were used as standards. D , immunoblot analysis for comparison of proliferation markers Ki67, pAkt, and total Akt, along with TLX, Oct-3/4, and GFAP differentiation marker in cells cultured under differentiating condition upon transfection of TLX singly or with Oct-3/4 shRNA ( Oct4 sh ) and TLX shRNA ( TLX sh ) alone or in combination with Oct-3/4 shRNA. GAPDH was used as loading control. E , immunoblot analysis for comparison of proliferation markers Ki67, pAkt, and total Akt, along with TLX, Oct-3/4 in cells cultured under differentiating conditions upon transfection of TLX, Oct-3/4 alone or in combination with TLX shRNA. GAPDH was used as loading control. F, table depicting the percentage of positive cells for BrdU staining in normoxia upon TLX overexpression ( TLX ) with or without Oct-3/4 depletion ( TLX + Oct Sh ) and hypoxia upon TLX depletion alone ( TLX sh ) or in combination with Oct-3/4 knockdown ( TLX sh + Oct Sh ).

    Journal: The Journal of Biological Chemistry

    Article Title: Nuclear Orphan Receptor TLX Induces Oct-3/4 for the Survival and Maintenance of Adult Hippocampal Progenitors upon Hypoxia *

    doi: 10.1074/jbc.M110.167445

    Figure Lengend Snippet: TLX is essential for Oct-3/4 expression in hypoxia. A , immunoblot analysis for TLX and Oct-3/4 in AHP cells cultured in the proliferating ( FGF +) or differentiating conditions ( FGF −) upon different time intervals of hypoxia. GAPDH was used as loading control. B , immunofluorescence analysis for TLX ( green ) and Oct-3/4 ( red ) in AHP cells cultured in the presence or absence of FGF under normoxia or 24 h hypoxia. C , comparison of TLX, Oct-3/4, GFAP, and Map2a protein levels in AHP cells cultured in proliferating conditions and depleted of TLX by shRNA ( Sh TLX ), or differentiating conditions, overexpressing TLX. GAPDH was used as loading control. A control shRNA ( Sh Ctrl ) and the vector control ( Vec ctrl ) were used as standards. D , immunoblot analysis for comparison of proliferation markers Ki67, pAkt, and total Akt, along with TLX, Oct-3/4, and GFAP differentiation marker in cells cultured under differentiating condition upon transfection of TLX singly or with Oct-3/4 shRNA ( Oct4 sh ) and TLX shRNA ( TLX sh ) alone or in combination with Oct-3/4 shRNA. GAPDH was used as loading control. E , immunoblot analysis for comparison of proliferation markers Ki67, pAkt, and total Akt, along with TLX, Oct-3/4 in cells cultured under differentiating conditions upon transfection of TLX, Oct-3/4 alone or in combination with TLX shRNA. GAPDH was used as loading control. F, table depicting the percentage of positive cells for BrdU staining in normoxia upon TLX overexpression ( TLX ) with or without Oct-3/4 depletion ( TLX + Oct Sh ) and hypoxia upon TLX depletion alone ( TLX sh ) or in combination with Oct-3/4 knockdown ( TLX sh + Oct Sh ).

    Article Snippet: The cells were harvested; protein was separated on SDS gel, electroblotted onto a PVDF membrane, and incubated with 5% bovine serum albumin (BSA) in TBS with 0.1% Tween 20, and the membranes were probed with the monoclonal antibodies anti-GFAP (1:1000, Dako), anti-TLX (1:1000, Life Span Technologies), anti-Map2a (1:2000, Millipore), anti-GAPDH (1:2000, Sigma), anti-Ηif2α (1:1000), anti-pAkt (1:1000), anti-total Akt (1:1000), anti-Ki67 (1:2000, Millipore), and anti-Oct-3/4 (1:1000, Santa Cruz Biotechnology).

    Techniques: Expressing, Cell Culture, Immunofluorescence, shRNA, Plasmid Preparation, Marker, Transfection, BrdU Staining, Over Expression

    Maternal undernutrition associated with ZIKV during pregnancy results in neurodevelopmental impairments 3 days after infection (E15). ( A ) Quantification of body weight of embryos at E15 [Kruskal-Wallis, 8.360 (*); multiple comparisons with Mann-Whitney U : Co/Mock versus LP/ZIKV, Z = −1.959 (*)]. Variance is significantly different between groups, as estimated by Bartlett test K -squared, 19.929 (***). ( B ) Analysis of microglia/macrophage recruitment with Iba1 + immunostaining (red) reveals the presence of reactive cells in the developing brain of LP/ZIKV group, in particular, in the lateral ventricles. ( C ) Immunostaining and quantification of cycling cells in the proliferative zone show a reduction of Ki67 + cells (green) in LP/ZIKV cortex. Analysis of variance (ANOVA) F = 3.838 (*); least significant difference (LSD): Co/Mock versus LP/ZIKV (*) ( P -adj > 0.05), Co/ZIKV versus LP/ZIKV (*) ( P -adj > 0.05), LP/Mock versus LP/ZIKV (*) ( P -adj > 0.05), df = 3. DAPI, 4′-6-diamidino-2-phenylindole. ( D ) Immunostaining and quantification of early-born neurons labeled with CTIP2 + (green). LP/ZIKV results in a reduction in cortical neurons compared with controls. ANOVA F = 4.77 (*); LSD: Co/ZIKV versus LP/ZIKV (*) ( P -adj > 0.05), LP/Mock versus LP/ZIKV (**) [ P -adj

    Journal: Science Advances

    Article Title: Congenital Zika syndrome is associated with maternal protein malnutrition

    doi: 10.1126/sciadv.aaw6284

    Figure Lengend Snippet: Maternal undernutrition associated with ZIKV during pregnancy results in neurodevelopmental impairments 3 days after infection (E15). ( A ) Quantification of body weight of embryos at E15 [Kruskal-Wallis, 8.360 (*); multiple comparisons with Mann-Whitney U : Co/Mock versus LP/ZIKV, Z = −1.959 (*)]. Variance is significantly different between groups, as estimated by Bartlett test K -squared, 19.929 (***). ( B ) Analysis of microglia/macrophage recruitment with Iba1 + immunostaining (red) reveals the presence of reactive cells in the developing brain of LP/ZIKV group, in particular, in the lateral ventricles. ( C ) Immunostaining and quantification of cycling cells in the proliferative zone show a reduction of Ki67 + cells (green) in LP/ZIKV cortex. Analysis of variance (ANOVA) F = 3.838 (*); least significant difference (LSD): Co/Mock versus LP/ZIKV (*) ( P -adj > 0.05), Co/ZIKV versus LP/ZIKV (*) ( P -adj > 0.05), LP/Mock versus LP/ZIKV (*) ( P -adj > 0.05), df = 3. DAPI, 4′-6-diamidino-2-phenylindole. ( D ) Immunostaining and quantification of early-born neurons labeled with CTIP2 + (green). LP/ZIKV results in a reduction in cortical neurons compared with controls. ANOVA F = 4.77 (*); LSD: Co/ZIKV versus LP/ZIKV (*) ( P -adj > 0.05), LP/Mock versus LP/ZIKV (**) [ P -adj

    Article Snippet: After standard antigenic retrieval, the following primary antibodies were incubated overnight: mouse J2 anti-dsRNA I (1:500), rat anti-CTIP2 (1:500; Abcam, ab18465), rabbit anti-Ki67 (1:100; EMD Millipore, AB9260), mouse anti-Iba1 (1:200; EMD Millipore, MABN92), rabbit anti-Iba1 (1:500 Wako Chemicals USA Inc.), and rabbit anti-CD31 (Abcam, ab28364).

    Techniques: Infection, MANN-WHITNEY, Immunostaining, Labeling

    Cell proliferation in the hippocampal SGZ is upregulated by acute restraint stress. (A) To compare the extent of cell proliferation, sectioned brain samples were immunostained with Ki67 (green) and mature neuronal marker, NeuN, antibodies (red). (B) Quantification of Ki67-positive cells in acute RST stress mice and the control group. Although extended subjection to restraint stress did not significantly change the number of proliferating cells, RST stress upregulated the number of Ki67-positive cells in the SGZ. * P

    Journal: Laboratory Animal Research

    Article Title: Repeated restraint stress promotes hippocampal neuronal cell ciliogenesis and proliferation in mice

    doi: 10.5625/lar.2018.34.4.203

    Figure Lengend Snippet: Cell proliferation in the hippocampal SGZ is upregulated by acute restraint stress. (A) To compare the extent of cell proliferation, sectioned brain samples were immunostained with Ki67 (green) and mature neuronal marker, NeuN, antibodies (red). (B) Quantification of Ki67-positive cells in acute RST stress mice and the control group. Although extended subjection to restraint stress did not significantly change the number of proliferating cells, RST stress upregulated the number of Ki67-positive cells in the SGZ. * P

    Article Snippet: The primary antibodies used were rabbit anti-AC (polyclonal) (1:1000; Santa Cruz), mouse anti-GFAP (1:500; Sigma), mouse anti-Nestin (1:400; Abcam), mouse anti-NeuN (1:500; Millipore), and rabbit anti-Ki67 (1:500; Abcam).

    Techniques: Marker, Mouse Assay

    Combination Epigenetic Treatment Reduces Lung Tumor Burden and Progression in Mouse Models of NSCLC (A) Representative H E-stained images of lung sections from mice treated with mock or Aza + ITF-2357. Scale bar, 100 μm. (B) Quantitation of total tumor area occupied by lesions in lungs of LSL-Kras G12D mice treated with mock or Aza + ITF-2357. Data are presented as mean ± SEM (p value determined by two-tailed t test; n = 6 mock and n = 7 Aza + ITF-2357 mice per group/2 sections analyzed per mouse). (C) Representative Ki67-stained IHC images of lung sections from mice treated with mock or Aza + ITF-2357 (n = 5 per group). Scale bar, 100 μm. (D) Lewis Lung Carcinoma (LLC) tumor weights of subcutaneous explants from 1-month mock- and Aza + ITF-2357-treated mice (n = 19 mice per group; error bars, SEM). (E) Representative H E-stained images of lung metastasis from LLC mice obtained from 1-month mock- and Aza + ITF-2357-treated mice (n = 12 mice per group), with the indicated percentage frequency of metastasis. (F) Volcano plot of relative RNA expression from LSL-Kras G12D mouse lung tumors treated for 3 months with Aza + ITF-2357 as compared to mock mice. Genes in upper left and right quadrants are significantly differentially expressed (microarray, n = 2 per group). (G) GSEA (KEGG, REACTOME, and HALLMARK) pathway distribution for Aza + ITF-2357 versus mock tumors from LSL-Kras G12D mice. Horizontal line denotes FDR significance cutoff of 0.25. Immune- and cell cycle-related gene sets are demarcated by green and red dots, respectively (microarray, n = 2 per group). (H) Gene sets upregulated in LSL-Kras G12D mice (FDR

    Journal: Cell

    Article Title: Epigenetic Therapy Ties MYC Depletion to Reversing Immune Evasion and Treating Lung Cancer

    doi: 10.1016/j.cell.2017.10.022

    Figure Lengend Snippet: Combination Epigenetic Treatment Reduces Lung Tumor Burden and Progression in Mouse Models of NSCLC (A) Representative H E-stained images of lung sections from mice treated with mock or Aza + ITF-2357. Scale bar, 100 μm. (B) Quantitation of total tumor area occupied by lesions in lungs of LSL-Kras G12D mice treated with mock or Aza + ITF-2357. Data are presented as mean ± SEM (p value determined by two-tailed t test; n = 6 mock and n = 7 Aza + ITF-2357 mice per group/2 sections analyzed per mouse). (C) Representative Ki67-stained IHC images of lung sections from mice treated with mock or Aza + ITF-2357 (n = 5 per group). Scale bar, 100 μm. (D) Lewis Lung Carcinoma (LLC) tumor weights of subcutaneous explants from 1-month mock- and Aza + ITF-2357-treated mice (n = 19 mice per group; error bars, SEM). (E) Representative H E-stained images of lung metastasis from LLC mice obtained from 1-month mock- and Aza + ITF-2357-treated mice (n = 12 mice per group), with the indicated percentage frequency of metastasis. (F) Volcano plot of relative RNA expression from LSL-Kras G12D mouse lung tumors treated for 3 months with Aza + ITF-2357 as compared to mock mice. Genes in upper left and right quadrants are significantly differentially expressed (microarray, n = 2 per group). (G) GSEA (KEGG, REACTOME, and HALLMARK) pathway distribution for Aza + ITF-2357 versus mock tumors from LSL-Kras G12D mice. Horizontal line denotes FDR significance cutoff of 0.25. Immune- and cell cycle-related gene sets are demarcated by green and red dots, respectively (microarray, n = 2 per group). (H) Gene sets upregulated in LSL-Kras G12D mice (FDR

    Article Snippet: Sections with primary antibodies Ki67 (Cell signaling, 9101, 1:500) were incubated at room temperature for 45 minutes.

    Techniques: Staining, Mouse Assay, Quantitation Assay, Significance Assay, Two Tailed Test, Immunohistochemistry, RNA Expression, Microarray

    Proliferation potential of Tcf21 lineage–traced fibroblasts residing in stable scar. ( A ) Experimental scheme whereby Tcf21 MCM/+ ; R26 EGFP mice previously treated with tamoxifen were subjected to MI and then treated with Ang II/PE through osmotic pump 4 weeks after MI. Mice were treated with EdU through daily i.p. injections for 6 days starting at day 2 after pump implantation, and hearts were harvested 4 hours after the last EdU injection for IHC analysis. ( B and C ) Quantification ( B ) and representative IHC images from 3 hearts analyzed ( C ) of EdU + (white) and Ki67 + (red) Tcf21 lineage–traced (EGFP + ) fibroblasts in the infarct region and septum of hearts from Tcf21 MCM/+ ; R26 EGFP mice that received treatment as shown in A . Nuclei are shown with DAPI (blue). Scale bars: 20 μm. ( D and E ) Quantification ( D ) and representative immunocytochemistry from 3 separate experiments ( E ) of EdU + (white) Tcf21 lineage–traced (EGFP + ) fibroblasts isolated from uninjured hearts and the infarct region of hearts 4 weeks later. EdU was given for 6 hours with and without TGF-β stimulation. Scale bars: 200 μm. ( F ) Representative immunocytochemistry images from 3 separate experiments showing αSMA stress fibers (red) in Tcf21 lineage–traced fibroblasts isolated from uninjured hearts and the infarct region of hearts 4 weeks after MI. Cells were also treated with TGF-β for 3 days. Scale bars: 10 μm. ( B and D ) Data are shown as mean ± SD ( n = 3). ** P

    Journal: The Journal of Clinical Investigation

    Article Title: Specialized fibroblast differentiated states underlie scar formation in the infarcted mouse heart

    doi: 10.1172/JCI98215

    Figure Lengend Snippet: Proliferation potential of Tcf21 lineage–traced fibroblasts residing in stable scar. ( A ) Experimental scheme whereby Tcf21 MCM/+ ; R26 EGFP mice previously treated with tamoxifen were subjected to MI and then treated with Ang II/PE through osmotic pump 4 weeks after MI. Mice were treated with EdU through daily i.p. injections for 6 days starting at day 2 after pump implantation, and hearts were harvested 4 hours after the last EdU injection for IHC analysis. ( B and C ) Quantification ( B ) and representative IHC images from 3 hearts analyzed ( C ) of EdU + (white) and Ki67 + (red) Tcf21 lineage–traced (EGFP + ) fibroblasts in the infarct region and septum of hearts from Tcf21 MCM/+ ; R26 EGFP mice that received treatment as shown in A . Nuclei are shown with DAPI (blue). Scale bars: 20 μm. ( D and E ) Quantification ( D ) and representative immunocytochemistry from 3 separate experiments ( E ) of EdU + (white) Tcf21 lineage–traced (EGFP + ) fibroblasts isolated from uninjured hearts and the infarct region of hearts 4 weeks later. EdU was given for 6 hours with and without TGF-β stimulation. Scale bars: 200 μm. ( F ) Representative immunocytochemistry images from 3 separate experiments showing αSMA stress fibers (red) in Tcf21 lineage–traced fibroblasts isolated from uninjured hearts and the infarct region of hearts 4 weeks after MI. Cells were also treated with TGF-β for 3 days. Scale bars: 10 μm. ( B and D ) Data are shown as mean ± SD ( n = 3). ** P

    Article Snippet: Anti-Ki67 antibody (catalog 9129S) was purchased from Cell Signaling Technology.

    Techniques: Mouse Assay, Injection, Immunohistochemistry, Immunocytochemistry, Isolation

    Proliferation of Tcf21 lineage–traced fibroblasts after MI. ( A ) Schematic of the Tcf21 genetic locus with a tamoxifen-regulated MerCreMer cDNA cassette inserted into exon 1 (E1).The MerCreMer-containing Tcf21 locus was introduced into R26 EGFP mice containing a loxP site–flanked stop cassette upstream of EGFP to allow for Cre-dependent lineage tracing. ( B ) Experimental scheme whereby Tcf21 MCM/+ ; R26 EGFP mice were given tamoxifen for 4 weeks and rested for 1 week before MI surgery. Mice were treated with a single EdU injection at the indicated time points after MI, and hearts were harvested 4 hours after each EdU injection for IHC analysis. ( C – E ) Quantification of EdU + (white) and Ki67 + (red) Tcf21 lineage–traced (EGFP + ) fibroblasts (green) in infarct region ( C ) and border zone ( D ) after a single EdU injection at the indicated time points after MI by IHC and representative IHC images. The white bar in C represents a 0 time point. ( E ) Nuclei are shown with DAPI (blue); these same images are shown in Supplemental Figure 1C in a larger temporal array. ( F ) Experimental scheme of tamoxifen treatment of Tcf21 MCM/+ ; R26 EGFP mice before MI surgery and 7 daily EdU injections during indicated time periods after MI. Hearts were harvested 4 hours after the last EdU injection for IHC analysis. ( G – I ) Quantification of EdU + (white) and Ki67 + (red) Tcf21 lineage–traced fibroblasts (green) in the infarct region ( G ) and border zone ( H ) after 7 daily EdU injections during the indicated time periods after MI by IHC and representative IHC images ( I ). Nuclei are shown with DAPI (blue). ( J ) Quantification of Tcf21 lineage–traced fibroblasts in the infarct region at the indicated time points by FACS. Density of cells is presented as the number of cells per mg of infarct tissue. ( C , D , G , H , and J ) Data are shown as mean ± SD ( n = 3). E and I show representative images from 3 separate hearts analyzed. Scale bars: 20 μm.

    Journal: The Journal of Clinical Investigation

    Article Title: Specialized fibroblast differentiated states underlie scar formation in the infarcted mouse heart

    doi: 10.1172/JCI98215

    Figure Lengend Snippet: Proliferation of Tcf21 lineage–traced fibroblasts after MI. ( A ) Schematic of the Tcf21 genetic locus with a tamoxifen-regulated MerCreMer cDNA cassette inserted into exon 1 (E1).The MerCreMer-containing Tcf21 locus was introduced into R26 EGFP mice containing a loxP site–flanked stop cassette upstream of EGFP to allow for Cre-dependent lineage tracing. ( B ) Experimental scheme whereby Tcf21 MCM/+ ; R26 EGFP mice were given tamoxifen for 4 weeks and rested for 1 week before MI surgery. Mice were treated with a single EdU injection at the indicated time points after MI, and hearts were harvested 4 hours after each EdU injection for IHC analysis. ( C – E ) Quantification of EdU + (white) and Ki67 + (red) Tcf21 lineage–traced (EGFP + ) fibroblasts (green) in infarct region ( C ) and border zone ( D ) after a single EdU injection at the indicated time points after MI by IHC and representative IHC images. The white bar in C represents a 0 time point. ( E ) Nuclei are shown with DAPI (blue); these same images are shown in Supplemental Figure 1C in a larger temporal array. ( F ) Experimental scheme of tamoxifen treatment of Tcf21 MCM/+ ; R26 EGFP mice before MI surgery and 7 daily EdU injections during indicated time periods after MI. Hearts were harvested 4 hours after the last EdU injection for IHC analysis. ( G – I ) Quantification of EdU + (white) and Ki67 + (red) Tcf21 lineage–traced fibroblasts (green) in the infarct region ( G ) and border zone ( H ) after 7 daily EdU injections during the indicated time periods after MI by IHC and representative IHC images ( I ). Nuclei are shown with DAPI (blue). ( J ) Quantification of Tcf21 lineage–traced fibroblasts in the infarct region at the indicated time points by FACS. Density of cells is presented as the number of cells per mg of infarct tissue. ( C , D , G , H , and J ) Data are shown as mean ± SD ( n = 3). E and I show representative images from 3 separate hearts analyzed. Scale bars: 20 μm.

    Article Snippet: Anti-Ki67 antibody (catalog 9129S) was purchased from Cell Signaling Technology.

    Techniques: Mouse Assay, Injection, Immunohistochemistry, FACS

    Meg3 knockdown promoted angiogenesis after ischemic stroke. a Representative micro-CT images show the revascularization in control, Meg3, sh-Ctrl, and sh-Meg3 groups 14 days post-MCAO. Bar graph shows quantitative analysis of micro-CT vessel volume in each group. b Representative images show CD31 staining at 14 and 28 days in control, Meg3, sh-Ctrl, or sh-Meg3 groups. Bar = 100 μm. Bar graph shows quantification of the microvessel density in each group ( n = 5/group). c Representative images show Ki67/CD31 staining in control, Meg3, or sh-Meg3 groups 14 days post-MCAO. Bar = 50 μm. Bar graph shows quantification of Ki67/CD31-poistive cells in the perifocal region in each group ( n = 5/group). * P

    Journal: Molecular Neurobiology

    Article Title: Downregulation of the Long Non-Coding RNA Meg3 Promotes Angiogenesis After Ischemic Brain Injury by Activating Notch Signaling

    doi: 10.1007/s12035-016-0270-z

    Figure Lengend Snippet: Meg3 knockdown promoted angiogenesis after ischemic stroke. a Representative micro-CT images show the revascularization in control, Meg3, sh-Ctrl, and sh-Meg3 groups 14 days post-MCAO. Bar graph shows quantitative analysis of micro-CT vessel volume in each group. b Representative images show CD31 staining at 14 and 28 days in control, Meg3, sh-Ctrl, or sh-Meg3 groups. Bar = 100 μm. Bar graph shows quantification of the microvessel density in each group ( n = 5/group). c Representative images show Ki67/CD31 staining in control, Meg3, or sh-Meg3 groups 14 days post-MCAO. Bar = 50 μm. Bar graph shows quantification of Ki67/CD31-poistive cells in the perifocal region in each group ( n = 5/group). * P

    Article Snippet: The primary antibodies used were as follows: rabbit anti-RFP (1:200; Abcam Inc., USA), mouse anti-PECAM-1 (1:50; Santa Cruz Biotechnology, Germany), rabbit anti-Ki67 (1:200; Cell signaling technology, USA).

    Techniques: Micro-CT, Staining

    Combination treatment is efficacious in vivo and correlates with decreased COX-2 expression. (A) L3.6pl cells were implanted subcutaneously and treatment was administered once daily via oral gavage for 10 days (shaded region) or until the group mean reached 1000 mm 3 (n = 5 per group). Tumors were harvested from a separate cohort on Day 7 (dotted line) for pharmacodynamic analysis. (B and C) Immunohistochemistry for Ki67 was performed and quantified (Immunoratio) in Figure B as a ratio between Ki67 stained nuclei and total nuclear area, while C shows representative images of treated tumors. (D) Heatmap generated from RPPA analysis of tumor lysates showing changes in protein expression. (E) RPPA results were verified via immunoblotting analysis for COX-2 and Pdcd4 expression. ** indicates p

    Journal: Molecular cancer therapeutics

    Article Title: Cyclooxygenase-2 Influences Response to Co-Targeting of MEK and CDK4/6 in a Subpopulation of Pancreatic Cancers

    doi: 10.1158/1535-7163.MCT-18-0082

    Figure Lengend Snippet: Combination treatment is efficacious in vivo and correlates with decreased COX-2 expression. (A) L3.6pl cells were implanted subcutaneously and treatment was administered once daily via oral gavage for 10 days (shaded region) or until the group mean reached 1000 mm 3 (n = 5 per group). Tumors were harvested from a separate cohort on Day 7 (dotted line) for pharmacodynamic analysis. (B and C) Immunohistochemistry for Ki67 was performed and quantified (Immunoratio) in Figure B as a ratio between Ki67 stained nuclei and total nuclear area, while C shows representative images of treated tumors. (D) Heatmap generated from RPPA analysis of tumor lysates showing changes in protein expression. (E) RPPA results were verified via immunoblotting analysis for COX-2 and Pdcd4 expression. ** indicates p

    Article Snippet: The Ki67 antibody was obtained from Cell Signaling Technology.

    Techniques: In Vivo, Expressing, Immunohistochemistry, Staining, Generated

    Fatostatin inhibits growth of and induces EnRS in MCF-7 xenograft tumors. MCF-7 cell xenograft tumors were initiated in athymic mice supplemented with estradiol capsules. Once tumors reached ~25 mm 2 , FS or DMSO control were administered daily ( n = 12–14 tumors/group). a Tumor size was measured and plotted over time. b Tumors were excised after 16 days of treatment and weighed. Animal body weight on day 16 is indicated. c–e Ki67 and cleaved PARP were examined in FFPE tumor sections by IHC and quantified. Bars represent 100 µm. f p-eIF2α was examined by IF with DAPI as a nuclear stain in DMSO and FS-treated tumors. g Quantitation of immunofluorescence was performed using the cell seed/spot segmentation analysis in ImageJ FIJI. The number of cells with color intensity +5% over background were counted as positive staining for p-eIF2α. h The mean color intensity of each cell staining positive for p-eIF2α was determined and plotted as number of cells vs. intensity. i SREBP1 was examined by IF with DAPI as a nuclear stain in DMSO and FS-treated tumors. j The number of nuclei with SREBP1 staining was determined and plotted per 100 cells. * P

    Journal: Oncogenesis

    Article Title: Fatostatin induces pro- and anti-apoptotic lipid accumulation in breast cancer

    doi: 10.1038/s41389-018-0076-0

    Figure Lengend Snippet: Fatostatin inhibits growth of and induces EnRS in MCF-7 xenograft tumors. MCF-7 cell xenograft tumors were initiated in athymic mice supplemented with estradiol capsules. Once tumors reached ~25 mm 2 , FS or DMSO control were administered daily ( n = 12–14 tumors/group). a Tumor size was measured and plotted over time. b Tumors were excised after 16 days of treatment and weighed. Animal body weight on day 16 is indicated. c–e Ki67 and cleaved PARP were examined in FFPE tumor sections by IHC and quantified. Bars represent 100 µm. f p-eIF2α was examined by IF with DAPI as a nuclear stain in DMSO and FS-treated tumors. g Quantitation of immunofluorescence was performed using the cell seed/spot segmentation analysis in ImageJ FIJI. The number of cells with color intensity +5% over background were counted as positive staining for p-eIF2α. h The mean color intensity of each cell staining positive for p-eIF2α was determined and plotted as number of cells vs. intensity. i SREBP1 was examined by IF with DAPI as a nuclear stain in DMSO and FS-treated tumors. j The number of nuclei with SREBP1 staining was determined and plotted per 100 cells. * P

    Article Snippet: Antibodies against Ki67 and p-eIF2α (#9027S; #3398S) were purchased from Cell Signaling Technology.

    Techniques: Mouse Assay, Formalin-fixed Paraffin-Embedded, Immunohistochemistry, Staining, Quantitation Assay, Immunofluorescence

    In vivo potential tumorigenesis assay design, as well as percent survival and Ki67 expression of late-passage hNPCs and their neuronal progeny. ( A ) Experimental timeline, wherein 3 month old NOD- scid mice were grafted with either hNPCs or MACS-purified neurons (N) in the striatum, and allowed to survive for 6 months. ( B,C ) Representative images of GFP+ surviving cells in mice 6 months after grafting with either hNPCs (scale bar = 10 μm) ( B ) or immature neurons (scale bar = 10 μm) ( C ). ( D ) Difference in overall donor cell survival between cell groups is not significant. ( E ) Migration of implanted cells was estimated on the basis of “migratory profiles characterized by fusiform-shaped cell bodies with single leading and/or trailing processes” (Zheng et al. 38 ). For both cell groups, the total number of GFP+ cells displaying a migratory morphology was counted per the total number of GFP+ cells. Both cell groups displayed migratory characteristics. ( F ) Proliferative status of both cell groups is very low, as indicated by Ki67 immuno-reactivity, particularly in the neuron group. (Ctx = cortex, Str = striatum) Means + s.e.m. P > 0.05, unpaired two-tailed t test.

    Journal: Scientific Reports

    Article Title: Transplantation of Defined Populations of Differentiated Human Neural Stem Cell Progeny

    doi: 10.1038/srep23579

    Figure Lengend Snippet: In vivo potential tumorigenesis assay design, as well as percent survival and Ki67 expression of late-passage hNPCs and their neuronal progeny. ( A ) Experimental timeline, wherein 3 month old NOD- scid mice were grafted with either hNPCs or MACS-purified neurons (N) in the striatum, and allowed to survive for 6 months. ( B,C ) Representative images of GFP+ surviving cells in mice 6 months after grafting with either hNPCs (scale bar = 10 μm) ( B ) or immature neurons (scale bar = 10 μm) ( C ). ( D ) Difference in overall donor cell survival between cell groups is not significant. ( E ) Migration of implanted cells was estimated on the basis of “migratory profiles characterized by fusiform-shaped cell bodies with single leading and/or trailing processes” (Zheng et al. 38 ). For both cell groups, the total number of GFP+ cells displaying a migratory morphology was counted per the total number of GFP+ cells. Both cell groups displayed migratory characteristics. ( F ) Proliferative status of both cell groups is very low, as indicated by Ki67 immuno-reactivity, particularly in the neuron group. (Ctx = cortex, Str = striatum) Means + s.e.m. P > 0.05, unpaired two-tailed t test.

    Article Snippet: For immunostaining, free-floating sections were blocked in a solution of 1X PBS with 0.1–0.3% Triton X-100 and 5% normal goat serum, and then incubated at 4 °C overnight in primary antibody cocktails containing the following, as needed: rabbit anti-GFAP (1:500; catalog #Z0334; Dako), rabbit anti-Ki67 (1:500; NCL-Ki67p; Novocastra), rabbit anti-Doublecortin (1:1,000; ab77450; Abcam).

    Techniques: In Vivo, Expressing, Mouse Assay, Magnetic Cell Separation, Purification, Migration, Two Tailed Test

    In vitro phenotypes of hNPCs differentiated in the neuroblast assay (NBA), followed by enrichment of the immature neuron subpopulation using MACS. ( A ) Overall experimental design. hNPC neurospheres were dissociated into single cells and plated in the NBA, generating a mixed population of neurons (N) and astrocytes ( A ). The neurons were then isolated from the astrocytes via MACS. Lastly, phenotyping was performed on the cells during the three major phases of the experiment. ( B–D ) Fluorescent images of immunocytochemical staining show hNPCs significantly lose expression of the proliferation marker Ki67 during the NBA. Enrichment of the neuron subgroup after the NBA, using MACS, reduces Ki67 expression still further (scale bar = 20 μm) (49,6-diamidino-2-phenylindole (DAPI), blue). ( E–G ) hNPCs significantly gain expression of the immature neuron marker DCX during the NBA. Enrichment of the neuron subgroup after the NBA yields an even higher percentage of DCX+ cells (scale bar = 20 μm) (49,6-diamidino-2-phenylindole (DAPI), blue). ( H–J ) Expression of the astrocyte marker GFAP increases slightly during differentiation, but MACS purification removes virtually all the percentage of GFAP+ cells (scale bar = 20 μm) (49,6-diamidino-2-phenylindole (DAPI), blue). ( K ) Quantification of immunolabeled cells indicates the differentiation of hNPCs into immature neurons during the NBA, and the subsequent purification of neurons made possible by MACS. Means + s.e.m. * P

    Journal: Scientific Reports

    Article Title: Transplantation of Defined Populations of Differentiated Human Neural Stem Cell Progeny

    doi: 10.1038/srep23579

    Figure Lengend Snippet: In vitro phenotypes of hNPCs differentiated in the neuroblast assay (NBA), followed by enrichment of the immature neuron subpopulation using MACS. ( A ) Overall experimental design. hNPC neurospheres were dissociated into single cells and plated in the NBA, generating a mixed population of neurons (N) and astrocytes ( A ). The neurons were then isolated from the astrocytes via MACS. Lastly, phenotyping was performed on the cells during the three major phases of the experiment. ( B–D ) Fluorescent images of immunocytochemical staining show hNPCs significantly lose expression of the proliferation marker Ki67 during the NBA. Enrichment of the neuron subgroup after the NBA, using MACS, reduces Ki67 expression still further (scale bar = 20 μm) (49,6-diamidino-2-phenylindole (DAPI), blue). ( E–G ) hNPCs significantly gain expression of the immature neuron marker DCX during the NBA. Enrichment of the neuron subgroup after the NBA yields an even higher percentage of DCX+ cells (scale bar = 20 μm) (49,6-diamidino-2-phenylindole (DAPI), blue). ( H–J ) Expression of the astrocyte marker GFAP increases slightly during differentiation, but MACS purification removes virtually all the percentage of GFAP+ cells (scale bar = 20 μm) (49,6-diamidino-2-phenylindole (DAPI), blue). ( K ) Quantification of immunolabeled cells indicates the differentiation of hNPCs into immature neurons during the NBA, and the subsequent purification of neurons made possible by MACS. Means + s.e.m. * P

    Article Snippet: For immunostaining, free-floating sections were blocked in a solution of 1X PBS with 0.1–0.3% Triton X-100 and 5% normal goat serum, and then incubated at 4 °C overnight in primary antibody cocktails containing the following, as needed: rabbit anti-GFAP (1:500; catalog #Z0334; Dako), rabbit anti-Ki67 (1:500; NCL-Ki67p; Novocastra), rabbit anti-Doublecortin (1:1,000; ab77450; Abcam).

    Techniques: In Vitro, Magnetic Cell Separation, Isolation, Staining, Expressing, Marker, Purification, Immunolabeling

    In vivo survival and phenotypes of hNPCs and their neuronal progeny transplanted in P-zero NSG mice. ( A) Overall experimental design. One group of mice was grafted with hNPCs, while the other group was grafted with NBA-generated hNPC progeny. Mice in both groups were sacrificed after 10 and 56 days. ( B,C ) Representative images of GFP+ surviving cells in mice grafted with either hNPCs ( B ) or immature neurons ( C ) 8 weeks after transplantation. ( D ) Quantification of surviving GFP+ cells in mice grafted with either hNPCs sacrificed 8 weeks after transplantation. The number of surviving GFP+ cells per section were not significantly different between hNPC and neuron grafts. ( E–J ) Fluorescent images of immunohistochemical staining, 8 weeks after transplantation, show GFP+/GFAP+ hNPCs to be upregulated in the hNPC ( E–G ) versus neuron ( H–J ) grafts (scale bar = 20 μm). ( K ) Quantification of donor cell phenotypes in the ipsilateral ventricle, cortex, hippocampus, RMS, olfactory bulb and cerebellum. While no difference in DCX frequency was seen between grafted cell groups 8 weeks after transplantation, the hNPC graft did yield significantly more GFP+/GFAP+ cells. ( L–R ) Likewise GFP+/Ki67+ cells are more observable in hNPC ( L–N ) versus neuron ( O – Q ) grafts, 10 days after transplantation (scale bar = 20 μm). Quantitatively, ( R ) GFP+/Ki67+ cells were more frequent in the hNPC grafts than in the neuron grafts 10 days after transplantation (**), but this difference gradually disappeared by 8 weeks post implant. (OB = olfactory bulb, Ctx = cortex) Means + s.e.m. ### P

    Journal: Scientific Reports

    Article Title: Transplantation of Defined Populations of Differentiated Human Neural Stem Cell Progeny

    doi: 10.1038/srep23579

    Figure Lengend Snippet: In vivo survival and phenotypes of hNPCs and their neuronal progeny transplanted in P-zero NSG mice. ( A) Overall experimental design. One group of mice was grafted with hNPCs, while the other group was grafted with NBA-generated hNPC progeny. Mice in both groups were sacrificed after 10 and 56 days. ( B,C ) Representative images of GFP+ surviving cells in mice grafted with either hNPCs ( B ) or immature neurons ( C ) 8 weeks after transplantation. ( D ) Quantification of surviving GFP+ cells in mice grafted with either hNPCs sacrificed 8 weeks after transplantation. The number of surviving GFP+ cells per section were not significantly different between hNPC and neuron grafts. ( E–J ) Fluorescent images of immunohistochemical staining, 8 weeks after transplantation, show GFP+/GFAP+ hNPCs to be upregulated in the hNPC ( E–G ) versus neuron ( H–J ) grafts (scale bar = 20 μm). ( K ) Quantification of donor cell phenotypes in the ipsilateral ventricle, cortex, hippocampus, RMS, olfactory bulb and cerebellum. While no difference in DCX frequency was seen between grafted cell groups 8 weeks after transplantation, the hNPC graft did yield significantly more GFP+/GFAP+ cells. ( L–R ) Likewise GFP+/Ki67+ cells are more observable in hNPC ( L–N ) versus neuron ( O – Q ) grafts, 10 days after transplantation (scale bar = 20 μm). Quantitatively, ( R ) GFP+/Ki67+ cells were more frequent in the hNPC grafts than in the neuron grafts 10 days after transplantation (**), but this difference gradually disappeared by 8 weeks post implant. (OB = olfactory bulb, Ctx = cortex) Means + s.e.m. ### P

    Article Snippet: For immunostaining, free-floating sections were blocked in a solution of 1X PBS with 0.1–0.3% Triton X-100 and 5% normal goat serum, and then incubated at 4 °C overnight in primary antibody cocktails containing the following, as needed: rabbit anti-GFAP (1:500; catalog #Z0334; Dako), rabbit anti-Ki67 (1:500; NCL-Ki67p; Novocastra), rabbit anti-Doublecortin (1:1,000; ab77450; Abcam).

    Techniques: In Vivo, Mouse Assay, Generated, Transplantation Assay, Immunohistochemistry, Staining

    CFC neural cultures show progenitor pool depletion, early maturation, and imbalance of neural cell types (A) Western blot analysis of the components of the Ras/MAPK pathway and its phosphorylation status, and quantification of the blots (B) from protein lysates extracted from CFC and control NPC cultures. The results were normalized with their corresponding α-tubulin values (assigned a value of 1) and are mean ±SD of two independent experiments (C) Double immunostaining for BrdU (green) and Ki67 (red) of early neuronal progenitors within 48h of differentiation; progenitors that exited the cell cycle, %(BrdU + Ki67 − )/BrDU + cells (orange arrows) were quantified. (D) Cleaved caspase 3 staining for cell death of the same NPCs and subsequent quantification. (E) Immunostaining of TUJ1, MAP2, NEUN, GABA, and TBR1 of CFC lines and control lines at week 1 of neural differentiation. Quantification of these stainings are shown as percentage of TUJ1 + cells (F), MAP2 + cells (G), NEUN + cells (H), average neurite length of TUJ1+ cells (I), average number of neurites per TUJ1 + cell (J), percentage of GABA + cells (K), and TBR1+ cells (L). (M) mRNA levels of MAP2 , deep layer markers TBR1 , CTIP2, and FEZF2 , upper layer markers CUX1 and SATB2 , and GABA precursors GAD1 and GAD2 in one-week old neural cultures. ΔCt values were calculated by normalizing the average Ct value of each cell line by the average Ct value of GUSB of the same cell line; ΔΔCt was calculated by normalizing the ΔCt value of each line to the average ΔCt value of the control lines. (N) Immunostaining of TUJ1, GFAP, CD44, S100, MAP2, FOXP2, and CUX1 at week 5 of neural differentiation. Quantification of these stainings is shown as a percentage of cells expressing GFAP (O), MAP2 (P), FOXP2 (Q) and CUX1 (R). (S) mRNA level analysis of astroglial cell markers GFAP , S100B , CD44 , neuronal marker MAP2 , deep layer markers TBR1 , CTIP2, and FEZF2 , upper layer markers CUX1 and SATB2 in five-week old neural cultures. ΔCt values were calculated by normalizing the average Ct value of each cell lines by the average Ct value of GUSB of the same cell line; ΔΔCt was calculated by normalizing the ΔCt value of each line to the average ΔCt value of the control lines. For all immunostainings depicted, at least three fields per coverslip from three independent experiments were counted. For all scatter plots illustrated, values represent mean±SD per condition, calculated with repeated measures ANOVA, accounting for biological and technical replicate values. Number of subclones and independent experiments performed are detailed in Supplementary Table 3 . Values for mean and standard deviation of each control and CFC subject for each experiment are detailed in Supplementary Table 7 . p ≤0.05 (*), p ≤0.01 (**), p ≤0.001 (***).

    Journal: Molecular psychiatry

    Article Title: Patient-derived iPSCs show premature neural differentiation and neuron-type specific phenotypes relevant to neurodevelopment

    doi: 10.1038/mp.2017.238

    Figure Lengend Snippet: CFC neural cultures show progenitor pool depletion, early maturation, and imbalance of neural cell types (A) Western blot analysis of the components of the Ras/MAPK pathway and its phosphorylation status, and quantification of the blots (B) from protein lysates extracted from CFC and control NPC cultures. The results were normalized with their corresponding α-tubulin values (assigned a value of 1) and are mean ±SD of two independent experiments (C) Double immunostaining for BrdU (green) and Ki67 (red) of early neuronal progenitors within 48h of differentiation; progenitors that exited the cell cycle, %(BrdU + Ki67 − )/BrDU + cells (orange arrows) were quantified. (D) Cleaved caspase 3 staining for cell death of the same NPCs and subsequent quantification. (E) Immunostaining of TUJ1, MAP2, NEUN, GABA, and TBR1 of CFC lines and control lines at week 1 of neural differentiation. Quantification of these stainings are shown as percentage of TUJ1 + cells (F), MAP2 + cells (G), NEUN + cells (H), average neurite length of TUJ1+ cells (I), average number of neurites per TUJ1 + cell (J), percentage of GABA + cells (K), and TBR1+ cells (L). (M) mRNA levels of MAP2 , deep layer markers TBR1 , CTIP2, and FEZF2 , upper layer markers CUX1 and SATB2 , and GABA precursors GAD1 and GAD2 in one-week old neural cultures. ΔCt values were calculated by normalizing the average Ct value of each cell line by the average Ct value of GUSB of the same cell line; ΔΔCt was calculated by normalizing the ΔCt value of each line to the average ΔCt value of the control lines. (N) Immunostaining of TUJ1, GFAP, CD44, S100, MAP2, FOXP2, and CUX1 at week 5 of neural differentiation. Quantification of these stainings is shown as a percentage of cells expressing GFAP (O), MAP2 (P), FOXP2 (Q) and CUX1 (R). (S) mRNA level analysis of astroglial cell markers GFAP , S100B , CD44 , neuronal marker MAP2 , deep layer markers TBR1 , CTIP2, and FEZF2 , upper layer markers CUX1 and SATB2 in five-week old neural cultures. ΔCt values were calculated by normalizing the average Ct value of each cell lines by the average Ct value of GUSB of the same cell line; ΔΔCt was calculated by normalizing the ΔCt value of each line to the average ΔCt value of the control lines. For all immunostainings depicted, at least three fields per coverslip from three independent experiments were counted. For all scatter plots illustrated, values represent mean±SD per condition, calculated with repeated measures ANOVA, accounting for biological and technical replicate values. Number of subclones and independent experiments performed are detailed in Supplementary Table 3 . Values for mean and standard deviation of each control and CFC subject for each experiment are detailed in Supplementary Table 7 . p ≤0.05 (*), p ≤0.01 (**), p ≤0.001 (***).

    Article Snippet: Neural cultures were processed for immunostaining with mouse anti-BrdU (1:100, BD Biosciences) and rabbit anti-Ki67 antibodies (1:400, Abcam).

    Techniques: Western Blot, Double Immunostaining, Staining, Immunostaining, Expressing, Marker, Standard Deviation

    Decreased phosphorylation of AKT is associated with progenitor cell pool depletion in CFC (A) Crosstalk between the Ras/Raf/MEK/ERK and Ras/PI3K/AKT pathway, and the targets of Wortmannin and SC79 (adapted from 64 ). (B) Double immunostaining for BrdU (red) and Ki67 (green) of early neuronal progenitors within 48h of differentiation in control NPCs, control NPCs pre-treated with Wortmannin, CFC NPCs, and CFC NPCs pre-treated with SC79; progenitors that exited the cell cycle, %(BrdU + Ki67 − )/BrdU + cells (orange arrows) were quantified and graphed in (C). (D) Immunostaining of MAP2 in control one-week old neural cultures, control one-week old neural cultures pre-treated with Wortmannin, CFC one-week old neural cultures, and CFC one-week old neural cultures pre-treated with SC79. (E) Quantification of the percentage of MAP2 positive DAPI in these stainings. (F) Immunostaining of TUJ1 in control one-week old neural cultures, control one-week old neural cultures pre-treated with Wortmannin, CFC one-week neural cultures, and CFC one-week old neural cultures pre-treated with SC79. Quantification of the average number of neurites per TUJ1 + cell (G). (H) Immunostaining of GFAP and TUJ1 in control five-week old neural cultures, control five-week old neural cultures pre-treated with Wortmannin, CFC five-week old neural cultures, and CFC five-week old neural cultures pre-treated with SC79. (I) Quantification of the percentage of GFAP positive DAPI in these stainings. (J) Immunostaining of MAP2 and FOXP2 in control five-week old neural cultures, control five-week old neural cultures pre-treated with Wortmannin, CFC five-week old neural cultures, and CFC five-week old neural cultures pre-treated with SC79. (K) Quantification of the percentage of FOXP2 positive DAPI in these stainings. (L) Immunostaining of MAP2 and CUX1 in control five-week old neural cultures, control five-week old neural cultures pre-treated with Wortmannin, CFC five-week old neural cultures, and CFC five-week old cultures pre-treated with SC79. (M) Quantification of the percentage of CUX1 positive DAPI in these stainings. For all immunostainings depicted, at least three fields per coverslip from three independent experiments were counted. For all scatter plots illustrated, values represent mean±SD per condition, calculated with repeated measures ANOVA, accounting for biological and technical replicates. Number of subclones and independent experiments performed are detailed in Supplementary Table 3 . Values for mean and standard deviation of each control and CFC subject for each experiment are detailed in Supplementary Table 7 . p ≤0.05 (*), p ≤0.01 (**), p ≤0.001 (***).

    Journal: Molecular psychiatry

    Article Title: Patient-derived iPSCs show premature neural differentiation and neuron-type specific phenotypes relevant to neurodevelopment

    doi: 10.1038/mp.2017.238

    Figure Lengend Snippet: Decreased phosphorylation of AKT is associated with progenitor cell pool depletion in CFC (A) Crosstalk between the Ras/Raf/MEK/ERK and Ras/PI3K/AKT pathway, and the targets of Wortmannin and SC79 (adapted from 64 ). (B) Double immunostaining for BrdU (red) and Ki67 (green) of early neuronal progenitors within 48h of differentiation in control NPCs, control NPCs pre-treated with Wortmannin, CFC NPCs, and CFC NPCs pre-treated with SC79; progenitors that exited the cell cycle, %(BrdU + Ki67 − )/BrdU + cells (orange arrows) were quantified and graphed in (C). (D) Immunostaining of MAP2 in control one-week old neural cultures, control one-week old neural cultures pre-treated with Wortmannin, CFC one-week old neural cultures, and CFC one-week old neural cultures pre-treated with SC79. (E) Quantification of the percentage of MAP2 positive DAPI in these stainings. (F) Immunostaining of TUJ1 in control one-week old neural cultures, control one-week old neural cultures pre-treated with Wortmannin, CFC one-week neural cultures, and CFC one-week old neural cultures pre-treated with SC79. Quantification of the average number of neurites per TUJ1 + cell (G). (H) Immunostaining of GFAP and TUJ1 in control five-week old neural cultures, control five-week old neural cultures pre-treated with Wortmannin, CFC five-week old neural cultures, and CFC five-week old neural cultures pre-treated with SC79. (I) Quantification of the percentage of GFAP positive DAPI in these stainings. (J) Immunostaining of MAP2 and FOXP2 in control five-week old neural cultures, control five-week old neural cultures pre-treated with Wortmannin, CFC five-week old neural cultures, and CFC five-week old neural cultures pre-treated with SC79. (K) Quantification of the percentage of FOXP2 positive DAPI in these stainings. (L) Immunostaining of MAP2 and CUX1 in control five-week old neural cultures, control five-week old neural cultures pre-treated with Wortmannin, CFC five-week old neural cultures, and CFC five-week old cultures pre-treated with SC79. (M) Quantification of the percentage of CUX1 positive DAPI in these stainings. For all immunostainings depicted, at least three fields per coverslip from three independent experiments were counted. For all scatter plots illustrated, values represent mean±SD per condition, calculated with repeated measures ANOVA, accounting for biological and technical replicates. Number of subclones and independent experiments performed are detailed in Supplementary Table 3 . Values for mean and standard deviation of each control and CFC subject for each experiment are detailed in Supplementary Table 7 . p ≤0.05 (*), p ≤0.01 (**), p ≤0.001 (***).

    Article Snippet: Neural cultures were processed for immunostaining with mouse anti-BrdU (1:100, BD Biosciences) and rabbit anti-Ki67 antibodies (1:400, Abcam).

    Techniques: Double Immunostaining, Immunostaining, Standard Deviation

    CFC cortical glutamatergic neuronal cultures show increased number of neurites per cell and decreased phosphorylation of BRAF (A) Double immunostaining for BrdU and Ki67 in early neuronal progenitors within 48h of differentiation with and without the addition of Compound E; progenitors that exited the cell cycle, %(BrdU + Ki67 − )/BrDU + cells were quantified. (B) Immunostaining of TUJ1 and TBR1 at week 1 of glutamatergic cortical neuron differentiation, and (E) of MAP2, TBR1, CUX1, and FOXP2 at week 5 of glutamatergic cortical neuron differentiation. Quantification of these staining is shown in (C–I). For the above immunostainings depicted, at least three fields per coverslip from three independent experiments were counted. (J) Fluorescent microscopy analysis of GFP-labelled five-week old control-derived and CFC-derived neurons. For the morphometric analysis, 20 GFP + TUJ1 + neurons per coverslip from three independent experiments were counted. Image J analysis of soma size (K), neurites per cell (L), neurite length (M), branches per neurite (N), branch length (O), and Sholl analysis of the number of intersecting neurites (P). Western blot analysis of the components of the Ras/MAPK pathway and its phosphorylation status (Q), and quantification of the blots (R) from protein lysates extracted from five-week old CFC and control glutamatergic cortical neuron cultures. The results were normalized with their corresponding α-tubulin values (assigned a value of 1) and are mean±SD of two independent experiments. For all immunostainings depicted, at least three fields per coverslip from three independent experiments were counted. For all scatter plots illustrated, values represent mean±SD per condition, calculated with repeated measures ANOVA, accounting for biological and technical replicates. Number of subclones and independent experiments performed are detailed in Supplementary Table 3 . Values for mean and standard deviation of each control and CFC subject for each experiment are detailed in Supplementary Table 7 . p ≤0.05 (*), p ≤0.01 (**), p ≤0.001 (***).

    Journal: Molecular psychiatry

    Article Title: Patient-derived iPSCs show premature neural differentiation and neuron-type specific phenotypes relevant to neurodevelopment

    doi: 10.1038/mp.2017.238

    Figure Lengend Snippet: CFC cortical glutamatergic neuronal cultures show increased number of neurites per cell and decreased phosphorylation of BRAF (A) Double immunostaining for BrdU and Ki67 in early neuronal progenitors within 48h of differentiation with and without the addition of Compound E; progenitors that exited the cell cycle, %(BrdU + Ki67 − )/BrDU + cells were quantified. (B) Immunostaining of TUJ1 and TBR1 at week 1 of glutamatergic cortical neuron differentiation, and (E) of MAP2, TBR1, CUX1, and FOXP2 at week 5 of glutamatergic cortical neuron differentiation. Quantification of these staining is shown in (C–I). For the above immunostainings depicted, at least three fields per coverslip from three independent experiments were counted. (J) Fluorescent microscopy analysis of GFP-labelled five-week old control-derived and CFC-derived neurons. For the morphometric analysis, 20 GFP + TUJ1 + neurons per coverslip from three independent experiments were counted. Image J analysis of soma size (K), neurites per cell (L), neurite length (M), branches per neurite (N), branch length (O), and Sholl analysis of the number of intersecting neurites (P). Western blot analysis of the components of the Ras/MAPK pathway and its phosphorylation status (Q), and quantification of the blots (R) from protein lysates extracted from five-week old CFC and control glutamatergic cortical neuron cultures. The results were normalized with their corresponding α-tubulin values (assigned a value of 1) and are mean±SD of two independent experiments. For all immunostainings depicted, at least three fields per coverslip from three independent experiments were counted. For all scatter plots illustrated, values represent mean±SD per condition, calculated with repeated measures ANOVA, accounting for biological and technical replicates. Number of subclones and independent experiments performed are detailed in Supplementary Table 3 . Values for mean and standard deviation of each control and CFC subject for each experiment are detailed in Supplementary Table 7 . p ≤0.05 (*), p ≤0.01 (**), p ≤0.001 (***).

    Article Snippet: Neural cultures were processed for immunostaining with mouse anti-BrdU (1:100, BD Biosciences) and rabbit anti-Ki67 antibodies (1:400, Abcam).

    Techniques: Double Immunostaining, Immunostaining, Staining, Microscopy, Derivative Assay, Western Blot, Standard Deviation

    Immunohistochemistry for Ki-67 in the dentate gyrus of the vehicle (A), scopolamine (SCO) (B), SCO+ vanillin (C), and SCO+4-hydroxybenzyl alcohol (4-HBA) (D) groups. In the vehicle group, Ki-67–immunoreactive cells (arrows) are shown in the subgranular zone. Ki-67-immunoreactive cells are significantly decreased in the SCO group; however, they are increased in the SCO+vanillin and SCO+4-HBA groups. GCL, granule cell layer; ML, molecular layer; PL, polymorphic layer. Scale bar=100 µm. (E) The mean number of Ki-67–immunoreactive cells per group (n=14 per group; * P

    Journal: Anatomy & Cell Biology

    Article Title: Vanillin and 4-hydroxybenzyl alcohol attenuate cognitive impairment and the reduction of cell proliferation and neuroblast differentiation in the dentate gyrus in a mouse model of scopolamine-induced amnesia

    doi: 10.5115/acb.2017.50.2.143

    Figure Lengend Snippet: Immunohistochemistry for Ki-67 in the dentate gyrus of the vehicle (A), scopolamine (SCO) (B), SCO+ vanillin (C), and SCO+4-hydroxybenzyl alcohol (4-HBA) (D) groups. In the vehicle group, Ki-67–immunoreactive cells (arrows) are shown in the subgranular zone. Ki-67-immunoreactive cells are significantly decreased in the SCO group; however, they are increased in the SCO+vanillin and SCO+4-HBA groups. GCL, granule cell layer; ML, molecular layer; PL, polymorphic layer. Scale bar=100 µm. (E) The mean number of Ki-67–immunoreactive cells per group (n=14 per group; * P

    Article Snippet: Immunohistochemistry As previously described [ ], immunohistochemical staining for neuronal nuclear antigen (NeuN, a marker for neurons), Ki-67 (a marker for proliferating cell) and doublecortin (DCX, a marker for neuroblast) was performed using rabbit anti-NeuN (1:1,000, Chemicon International, Temecula, CA, USA), rabbit anti-Ki-67 (1:100, Abcam, Cambridge, UK), or goat anti-DCX (1:100, Santa Cruz Biotechnology, Santa Cruz, CA, USA) as primary antibodies, and biotinylated goat anti-rabbit or rabbit anti-goat immunoglobulin G (1:200, Vector Laboratories, Burlingame, CA, USA) and streptavidin peroxidase complex (1:200, Vector Laboratories) as secondary antibodies.

    Techniques: Immunohistochemistry

    Effects of cold challenge or CL 316,243 treatment for 1 week or 4 weeks on the TBR-2 and Ki-67 positive cells in the dentate gyrus. Note that the TBR-2 and Ki-67 positive cells are abundant in the Cold1W and Cold4W groups when compared with that in the CON Cold1W and CON Cold 4W groups, respectively (A,C,E,G) . However, the CON CL1W, CL1W, CON CL4W, and CL4W groups show similar populations of TBR-2 and Ki-67 positive cells in the dentate gyrus (A,C,E,G) . GCL, granule cell layer; MoL, molecular layer; PoL, polymorphic layer. Quantitative analysis of TBR-2 and Ki-67 positive cells per section in the CON Cold1W, Cold1W, CON Cold4W, Cold4W, CON CL1W, CL1W, CON CL4W, and CL4W groups (B,D,F,H) ( n = 5 per group); ∗ indicates a significant difference when compared with the CON group ( p

    Journal: Frontiers in Neuroscience

    Article Title: Adult Hippocampal Neurogenesis Can Be Enhanced by Cold Challenge Independently From Beigeing Effects

    doi: 10.3389/fnins.2019.00092

    Figure Lengend Snippet: Effects of cold challenge or CL 316,243 treatment for 1 week or 4 weeks on the TBR-2 and Ki-67 positive cells in the dentate gyrus. Note that the TBR-2 and Ki-67 positive cells are abundant in the Cold1W and Cold4W groups when compared with that in the CON Cold1W and CON Cold 4W groups, respectively (A,C,E,G) . However, the CON CL1W, CL1W, CON CL4W, and CL4W groups show similar populations of TBR-2 and Ki-67 positive cells in the dentate gyrus (A,C,E,G) . GCL, granule cell layer; MoL, molecular layer; PoL, polymorphic layer. Quantitative analysis of TBR-2 and Ki-67 positive cells per section in the CON Cold1W, Cold1W, CON Cold4W, Cold4W, CON CL1W, CL1W, CON CL4W, and CL4W groups (B,D,F,H) ( n = 5 per group); ∗ indicates a significant difference when compared with the CON group ( p

    Article Snippet: They were then incubated with a rabbit anti-Ki-67 antibody (1:1,000; Abcam, Cambridge, United Kingdom) or goat anti-doublecortin (DCX) antibody (1:50; Santa Cruz Biotechnology, Santa Cruz, CA, United States) overnight at 25°C and, subsequently, treated with either a biotinylated goat anti-rabbit IgG or a rabbit anti-goat IgG, and a streptavidin-peroxidase complex (1:200; Vector Labs, Burlingame, CA, United States).

    Techniques:

    Loss of RNF220 inhibited NSC proliferation and promoted differentiation of NSCs. ( A – F ) Loss of RNF220 inhibited NSCs proliferation by EDU incorporation assay ( A – C ) and Ki67 immunostaining ( D – F ); the nuclei were marked by DAPI. Scale bar: 50 µm. ( C , F ) The quantification data for ( A , B , D , E ) respectively. ( G – L ) Immunofluorescence analyses of the differentiated neurons from WT and ZC4H2 −/− NSCs for β-tubulin III (TUJ1) ( G – I ) and MAP2 ( J – L ); the nuclei were marked by DAPI. Scale bar: 50 µm. ( I , L ) The quantification data for ( G , H , J , K ), respectively. ( M , N ) Expression of stemness marker genes (Nestin and Vimentin) in WT and RNF220 −/− NSCs. ( O , P ) Expression of neuronal marker genes (TUJ1 and MAP2) in differentiated WT and RNF220 −/− NSCs. β-actin was used as an internal control. ** p

    Journal: Cells

    Article Title: Loss of ZC4H2 and RNF220 Inhibits Neural Stem Cell Proliferation and Promotes Neuronal Differentiation

    doi: 10.3390/cells9071600

    Figure Lengend Snippet: Loss of RNF220 inhibited NSC proliferation and promoted differentiation of NSCs. ( A – F ) Loss of RNF220 inhibited NSCs proliferation by EDU incorporation assay ( A – C ) and Ki67 immunostaining ( D – F ); the nuclei were marked by DAPI. Scale bar: 50 µm. ( C , F ) The quantification data for ( A , B , D , E ) respectively. ( G – L ) Immunofluorescence analyses of the differentiated neurons from WT and ZC4H2 −/− NSCs for β-tubulin III (TUJ1) ( G – I ) and MAP2 ( J – L ); the nuclei were marked by DAPI. Scale bar: 50 µm. ( I , L ) The quantification data for ( G , H , J , K ), respectively. ( M , N ) Expression of stemness marker genes (Nestin and Vimentin) in WT and RNF220 −/− NSCs. ( O , P ) Expression of neuronal marker genes (TUJ1 and MAP2) in differentiated WT and RNF220 −/− NSCs. β-actin was used as an internal control. ** p

    Article Snippet: Primary antibodies used in this study are as follows: rabbit anti-Ki67 (Abcam, Cambridge, USA, Cat# ab15580), mouse anti-Nestin (Abcam, Cat# ab6142), rabbit anti-β-tubulin III (Biolegend, San Diego, CA, USA, Cat# PRB-435P), mouse anti-MAP2 (Sigma-Aldrich, St. Louis, MO, USA, Cat# M9942), and goat anti-Sox2 (Santa Cruz Biotechnology, Dallas, TX, USA, Cat# sc-17320).

    Techniques: Immunostaining, Immunofluorescence, Expressing, Marker

    Loss of ZC4H2 inhibited neural stem cell (NSC) proliferation and promoted differentiation of NSCs. ( A – C ) The size (diameter) of neurosphere between wild type (WT) NSCs ( A ) and ZC4H2 −/− NSCs ( B ). Scale bar: 400 µm. ( C ) The quantification data (diameter) for ( A , B ) [ 22 ]. ( D – I ) Loss of ZC4H2 inhibited NSC proliferation by EDU incorporation assay ( D – F ) and Ki67 immunostaining ( G – I ). The nuclei were marked by DAPI. Scale bar: 50 µm. ( F , I ) The quantification data (percentage of EDU/DAPI and Ki67/DAPI) for ( D , E , G , H ), respectively. ( J – O ) Immunofluorescence analyses of the differentiated neurons from WT and ZC4H2 −/− NSCs for β-tubulin III (TUJ1) ( J – L ) and MAP2 ( M – O ). ( L , O ) The quantification data (percentage of TUJ1/DAPI and MAP2/DAPI) for ( J , K , M , N ), respectively. ( P , Q ) Relative mRNA expression levels of stemness marker genes (Nestin and Vimentin) in WT and ZC4H2 −/− NSCs. ( R , S ) Relative mRNA expression levels of neuronal marker genes (TUJ1 and MAP2) in differentiated WT and ZC4H2 −/− NSCs. The β-actin was used as an internal control. ** p

    Journal: Cells

    Article Title: Loss of ZC4H2 and RNF220 Inhibits Neural Stem Cell Proliferation and Promotes Neuronal Differentiation

    doi: 10.3390/cells9071600

    Figure Lengend Snippet: Loss of ZC4H2 inhibited neural stem cell (NSC) proliferation and promoted differentiation of NSCs. ( A – C ) The size (diameter) of neurosphere between wild type (WT) NSCs ( A ) and ZC4H2 −/− NSCs ( B ). Scale bar: 400 µm. ( C ) The quantification data (diameter) for ( A , B ) [ 22 ]. ( D – I ) Loss of ZC4H2 inhibited NSC proliferation by EDU incorporation assay ( D – F ) and Ki67 immunostaining ( G – I ). The nuclei were marked by DAPI. Scale bar: 50 µm. ( F , I ) The quantification data (percentage of EDU/DAPI and Ki67/DAPI) for ( D , E , G , H ), respectively. ( J – O ) Immunofluorescence analyses of the differentiated neurons from WT and ZC4H2 −/− NSCs for β-tubulin III (TUJ1) ( J – L ) and MAP2 ( M – O ). ( L , O ) The quantification data (percentage of TUJ1/DAPI and MAP2/DAPI) for ( J , K , M , N ), respectively. ( P , Q ) Relative mRNA expression levels of stemness marker genes (Nestin and Vimentin) in WT and ZC4H2 −/− NSCs. ( R , S ) Relative mRNA expression levels of neuronal marker genes (TUJ1 and MAP2) in differentiated WT and ZC4H2 −/− NSCs. The β-actin was used as an internal control. ** p

    Article Snippet: Primary antibodies used in this study are as follows: rabbit anti-Ki67 (Abcam, Cambridge, USA, Cat# ab15580), mouse anti-Nestin (Abcam, Cat# ab6142), rabbit anti-β-tubulin III (Biolegend, San Diego, CA, USA, Cat# PRB-435P), mouse anti-MAP2 (Sigma-Aldrich, St. Louis, MO, USA, Cat# M9942), and goat anti-Sox2 (Santa Cruz Biotechnology, Dallas, TX, USA, Cat# sc-17320).

    Techniques: Immunostaining, Immunofluorescence, Expressing, Marker

    Switching on in situ ROS production in the skin activates stem cell proliferation in the hair follicle niche and promotes a transient proliferation of epidermal and dermal cells a) BrdU label retaining cells (LRC) quantification in the hair follicle bulge region. The mean + SE (n=4) is represented. b) Immunological detection of the Ki67 proliferation marker in the hair follicle bulge region. c) Dorsal skin histological sections stained for inmunohistochemical detection of Ki67 (left panels) or with Masson′s trichrome (middle panels) showing transient effects in the skin induced by mALA+Light treatments, including cell proliferation, hyperplasia in the epidermis (vertical bars), a significant increase in dermal cellularity (squares) and strong cornification (arrowheads). Squares indicate equivalent areas in which cell numbers were quantified. Right panels show the quantification of interfollicular epidermis (IFE) thickness and dermal cellularity in 10 histological fields. The mean + SD (n=3) is represented. In a) and b) representative confocal microscopy images (maximum projections) of tail skin whole-mounts are shown. Bars: 100 μm.

    Journal: The Journal of investigative dermatology

    Article Title: Photoactivation of ROS production in situ transiently activates cell proliferation in mouse skin and in the hair follicle stem cell niche promoting hair growth and wound healing

    doi: 10.1038/jid.2015.248

    Figure Lengend Snippet: Switching on in situ ROS production in the skin activates stem cell proliferation in the hair follicle niche and promotes a transient proliferation of epidermal and dermal cells a) BrdU label retaining cells (LRC) quantification in the hair follicle bulge region. The mean + SE (n=4) is represented. b) Immunological detection of the Ki67 proliferation marker in the hair follicle bulge region. c) Dorsal skin histological sections stained for inmunohistochemical detection of Ki67 (left panels) or with Masson′s trichrome (middle panels) showing transient effects in the skin induced by mALA+Light treatments, including cell proliferation, hyperplasia in the epidermis (vertical bars), a significant increase in dermal cellularity (squares) and strong cornification (arrowheads). Squares indicate equivalent areas in which cell numbers were quantified. Right panels show the quantification of interfollicular epidermis (IFE) thickness and dermal cellularity in 10 histological fields. The mean + SD (n=3) is represented. In a) and b) representative confocal microscopy images (maximum projections) of tail skin whole-mounts are shown. Bars: 100 μm.

    Article Snippet: Primary antibodies used were FITC-conjugated mouse monoclonal anti BrdU (Roche); rabbit monoclonal antibodies against Src, Phospho-Src (Tyr416), AKT, Phospho-AKT (Ser473), p38 MAPK, Phospho-p38 MAPK (Thr180/Tyr182), p44/42 MAPK, Phospho-p44/42 MAPK (Thr202/Tyr204), Phospho SAPK/JNK (Thr183/Tyr185) and gammaH2AX (Ser 139) and Lef1 (all from Cell Signaling Technologies); rabbit monoclonal anti Ki67 (Neo Markers); rabbit polyclonal anti Src (Abcam); mouse monoclonal anti β-catenin (BD); mouse monoclonal anti Active-β-catenin (Millipore); goat polyclonal anti-Proliferin 2 (Prl2c3) and anti-MPO (both from Santa Cruz Biotechnology); mouse monoclonal antibodies against PCNA (Calbiochem), tubulin and BrdU (both from Sigma).

    Techniques: In Situ, Marker, Staining, Confocal Microscopy

    Characterization of human primary glioma surgical samples . Extensive GFAP (A, left panel) expression was detected in primary glioma surgical sample frozen sections using immunofluorescent staining with a rabbit anti-GFAP antibody. (B, left panel) Numerous dividing cells were detected in the same surgical glioma sample using an anti-Ki67 antibody. Right panels in (A) and (B) are nuclear counterstaining for GFAP and Ki67 stained sections, respectively. (C) Demonstration of specificity of anti-L1 antibodies anti-cytoplasmic polyclonal NCAM-L1 (left panel) and anti-ectodomain monoclonal UJ127 (right panel) by western blot analysis. Human L1-expressing quail QT6 cells (QT6/hFL1) were used as positive controls (PC), untransfected QT6 cells were used as negative controls (NC), and plain QT6 cell culture media (M) was used as an additional negative control. (D) L1 expression was found in human primary gliomas surgical samples (sample numbers 10-15 and 17-20) by western blot analysis using UJ127 anti-L1 antibody. Transfected QT6/hFL1 cells were used as positive control and QT6 cells were used as a negative control. Gels were loaded with 10 μg total protein and probed for GAPDH as a loading control (see text). (E) Analysis of surgical samples 18-20 using anti-L1 antibody NCAM-L1. Same blot was used as for (D, right panel, 10 μg total protein/well). GAPDH was used as a loading control. (F) Analysis of glioma surgical samples for L1 protease ADAM10, revealing that all samples were positive predominantly for active ADAM10 (approx. 55 kDa). (G) Surgical samples # 7, 18, 19, and 20 were analyzed by western blot with a rabbit anti-NF-M antibody to detect neurofilament expression. Adult rat brain (RB) lysate was used as a positive control for NF-M staining. (H) Media from surgical sample cells grown in culture were analyzed by western blot for L1. Soluble L1 was detectable in media from sample # 21. Positive controls (PC) were cell lysates from QT6/hFL1 cells, and untransfected cell lysate was used as negative control (NC). Media from CHO cells transfected with an L1 ectodomain vector (CHO-L1ecto) were also used as a positive control for soluble L1. Media from untransfected CHO cells were used as a negative control.

    Journal: Cancer Cell International

    Article Title: Stimulation of glioma cell motility by expression, proteolysis, and release of the L1 neural cell recognition molecule

    doi: 10.1186/1475-2867-9-27

    Figure Lengend Snippet: Characterization of human primary glioma surgical samples . Extensive GFAP (A, left panel) expression was detected in primary glioma surgical sample frozen sections using immunofluorescent staining with a rabbit anti-GFAP antibody. (B, left panel) Numerous dividing cells were detected in the same surgical glioma sample using an anti-Ki67 antibody. Right panels in (A) and (B) are nuclear counterstaining for GFAP and Ki67 stained sections, respectively. (C) Demonstration of specificity of anti-L1 antibodies anti-cytoplasmic polyclonal NCAM-L1 (left panel) and anti-ectodomain monoclonal UJ127 (right panel) by western blot analysis. Human L1-expressing quail QT6 cells (QT6/hFL1) were used as positive controls (PC), untransfected QT6 cells were used as negative controls (NC), and plain QT6 cell culture media (M) was used as an additional negative control. (D) L1 expression was found in human primary gliomas surgical samples (sample numbers 10-15 and 17-20) by western blot analysis using UJ127 anti-L1 antibody. Transfected QT6/hFL1 cells were used as positive control and QT6 cells were used as a negative control. Gels were loaded with 10 μg total protein and probed for GAPDH as a loading control (see text). (E) Analysis of surgical samples 18-20 using anti-L1 antibody NCAM-L1. Same blot was used as for (D, right panel, 10 μg total protein/well). GAPDH was used as a loading control. (F) Analysis of glioma surgical samples for L1 protease ADAM10, revealing that all samples were positive predominantly for active ADAM10 (approx. 55 kDa). (G) Surgical samples # 7, 18, 19, and 20 were analyzed by western blot with a rabbit anti-NF-M antibody to detect neurofilament expression. Adult rat brain (RB) lysate was used as a positive control for NF-M staining. (H) Media from surgical sample cells grown in culture were analyzed by western blot for L1. Soluble L1 was detectable in media from sample # 21. Positive controls (PC) were cell lysates from QT6/hFL1 cells, and untransfected cell lysate was used as negative control (NC). Media from CHO cells transfected with an L1 ectodomain vector (CHO-L1ecto) were also used as a positive control for soluble L1. Media from untransfected CHO cells were used as a negative control.

    Article Snippet: To detect mitotic cells, a rabbit polyclonal anti-Ki67 antibody (cat. # ab15580; Abcam, Cambridge, MA) was used followed by Alexa Fluor-594 secondary antibody.

    Techniques: Expressing, Staining, Western Blot, Cell Culture, Negative Control, Transfection, Positive Control, Plasmid Preparation

    Some supporting cells in Ad.MT58A-infected utricles survive for weeks in culture after reentering the cell cycle. ( A ) Diagram depicting the BrdU labeling paradigm. BrdU was either included in the culture medium for the entire culture period after infection with adenovirus, or it was washed out at 5 days post-virus (DPV) and utricles were cultured for an additional 5 d in its absence. ( B–C ) Confocal images show Ad.MT58A-infected utricles (1×10 9 TU/mL) fixed at 10 DPV after being cultured with BrdU from 1–10 DPV (B) or 1–5 DPV (C). Scale bar for B–C, 100 µm. ( D ) Graph shows quantification of the number of BrdU-labeled cells in the sensory epithelium from utricles cultured as depicted in A (gray and white bars). Quantification of 5 DPV BrdU labeling (same as in Fig. 5G) is shown to visualize the decline in BrdU-labeled cells from 5 DPV (black bars) to 10 DPV. Data shown is from co-infection experiments (OKSM), infection with 2×10 8 TU/mL Ad.MT58A (1×M), and infection with 1×10 9 TU/mL Ad.MT58A (5×M). ( E ) Confocal image of an Ad.MT58A-infected utricle (1×10 9 TU/mL) fixed at 21 DPV after being cultured with BrdU from 1–5 DPV. Antibody labeling for BrdU and Ki-67 is shown in green and red, respectively. Scale bar, 100 µm. ( F ) Confocal image of an Ad.MT58A-infected utricle (1×10 9 TU/mL) fixed at 21 DPV after being cultured with BrdU from 18–21 DPV. Antibody labeling for BrdU and myosin VIIA is shown in green and magenta, respectively. Scale bar, 100 µm. ( G ) Confocal image of an Ad.MT58A-infected utricle fixed at 10 DPV after switching from growth medium to differentiation medium at 5 DPV. BrdU (green) was included in the medium throughout. Scale bar, 100 µm. ( H ) Graph shows the mean number of BrdU-positive nuclei per sensory epithelium at 5, 10, and 14 DPV for the experiment described in G. White dashed lines demarcate the borders of the sensory epithelium in all panels.

    Journal: PLoS ONE

    Article Title: MYC Gene Delivery to Adult Mouse Utricles Stimulates Proliferation of Postmitotic Supporting Cells In Vitro

    doi: 10.1371/journal.pone.0048704

    Figure Lengend Snippet: Some supporting cells in Ad.MT58A-infected utricles survive for weeks in culture after reentering the cell cycle. ( A ) Diagram depicting the BrdU labeling paradigm. BrdU was either included in the culture medium for the entire culture period after infection with adenovirus, or it was washed out at 5 days post-virus (DPV) and utricles were cultured for an additional 5 d in its absence. ( B–C ) Confocal images show Ad.MT58A-infected utricles (1×10 9 TU/mL) fixed at 10 DPV after being cultured with BrdU from 1–10 DPV (B) or 1–5 DPV (C). Scale bar for B–C, 100 µm. ( D ) Graph shows quantification of the number of BrdU-labeled cells in the sensory epithelium from utricles cultured as depicted in A (gray and white bars). Quantification of 5 DPV BrdU labeling (same as in Fig. 5G) is shown to visualize the decline in BrdU-labeled cells from 5 DPV (black bars) to 10 DPV. Data shown is from co-infection experiments (OKSM), infection with 2×10 8 TU/mL Ad.MT58A (1×M), and infection with 1×10 9 TU/mL Ad.MT58A (5×M). ( E ) Confocal image of an Ad.MT58A-infected utricle (1×10 9 TU/mL) fixed at 21 DPV after being cultured with BrdU from 1–5 DPV. Antibody labeling for BrdU and Ki-67 is shown in green and red, respectively. Scale bar, 100 µm. ( F ) Confocal image of an Ad.MT58A-infected utricle (1×10 9 TU/mL) fixed at 21 DPV after being cultured with BrdU from 18–21 DPV. Antibody labeling for BrdU and myosin VIIA is shown in green and magenta, respectively. Scale bar, 100 µm. ( G ) Confocal image of an Ad.MT58A-infected utricle fixed at 10 DPV after switching from growth medium to differentiation medium at 5 DPV. BrdU (green) was included in the medium throughout. Scale bar, 100 µm. ( H ) Graph shows the mean number of BrdU-positive nuclei per sensory epithelium at 5, 10, and 14 DPV for the experiment described in G. White dashed lines demarcate the borders of the sensory epithelium in all panels.

    Article Snippet: Immunocytochemistry The following antibodies were used: rabbit anti-myosin VIIA (1∶200; Proteus Biosciences, Ramona, CA; # 25-6790) and mouse anti-myosin VIIA (1∶100; Developmental Studies Hybridoma Bank, Iowa City, Iowa; # MYO7A 138-1) to label hair cell soma; mouse anti-BrdU (1∶50; BD Biosciences; # 347580) to label cells that had incorporated BrdU during S-phase; rabbit anti-Ki67 (1∶200; Thermofisher Scientific, Kalamazoo, MI; # RM-9106-S0) to label cells in the active G1, S, G2, and M phases of the cell cycle; mouse anti-phospho-histone H3 (Ser10) (PH3-Ser10; 1∶200, Cell Signaling Technology, Danvers, MA; # 9706) to label cells in M phase; rabbit anti-Oct3/4 (1∶200; Santa Cruz Biotechnology, Santa Cruz, California; # SC-5279); mouse anti-Klf4 (1∶200; Abcam, Cambridge MA; # AB75486); rabbit anti-Sox2 (1∶200; Millipore, Billerica, MA; # AB5603); mouse anti-c-Myc (1∶200; Santa Cruz Biotechnology; # SC-40); mouse anti-HA (1∶200; Abcam; # AB18181); and rabbit anti-activated-caspase 3 to label cells undergoing apoptosis (1∶200; Abcam; # AB3623).

    Techniques: Infection, Labeling, Cell Culture, Antibody Labeling

    Supporting cells that reenter the cell cycle after Ad.MT58A infection can progress to mitosis. ( A ) Confocal images show an Ad.MT58A-infected utricle (1×10 9 TU/mL) that was fixed at 7 days post-virus (DPV) and co-labeled with antibodies to BrdU (red) and Ki-67 (green). Phalloidin labeling (grayscale) is shown to aid in visualizing the borders of the sensory epithelium. White dashed lines demarcate the borders of the sensory epithelium. Scale bar, 100 µm. Insets show high-resolution views of nuclei in the sensory epithelium. Arrows indicate nuclei that labeled with antibodies to BrdU but not Ki67. Scale bar for insets, 5 µm. ( B ) Graph shows the mean number of BrdU-labeled nuclei (green data points) and Ki-67-labeled nuclei (red data points) per sensory epithelium at 5, 7, and 10 DPV. ( C ) Graph shows quantification of the percentage of the BrdU-positive population that did not label with Ki67 antibodies (green data points) and the percentage of the Ki-67-positive population that did not label with BrdU antibodies (red data points). ( D ) Confocal image of an adult mouse utricle infected with Ad.MT58A (1×10 9 TU/mL) that was fixed at 7 DPV and co-labeled with antibodies to PH3-Ser10 (white) and Ki-67 (red). ( E ) Confocal images of a utricle from an embryonic day 17.5 (E17.5) mouse that was fixed in vivo and co-labeled with antibodies to PH3-Ser10 (white) and Ki-67 (red). Phalloidin labeling (grayscale) is shown to aid in visualizing the borders of the sensory epithelium. White dashed lines demarcate the borders of the sensory epithelium, and arrows in D and E indicate PH3-Ser10/Ki-67 co-labeled nuclei. Scale bar for D–E, 100 µm. ( F ) Graph shows quantification of the percentage of the Ki-67-positive populations that labeled with antibodies to PH3-Ser10. The difference in the percentage of PH3-Ser10-positive/Ki-67-positive nuclei did not reach statistical significance (p > 0.05; Student's t-test). The numbers above the gray bars indicate the mean number of PH3-Ser10-labeled nuclei per sensory epithelium. There were significantly more PH3-Ser10-positive nuclei in E17.5 utricles (p

    Journal: PLoS ONE

    Article Title: MYC Gene Delivery to Adult Mouse Utricles Stimulates Proliferation of Postmitotic Supporting Cells In Vitro

    doi: 10.1371/journal.pone.0048704

    Figure Lengend Snippet: Supporting cells that reenter the cell cycle after Ad.MT58A infection can progress to mitosis. ( A ) Confocal images show an Ad.MT58A-infected utricle (1×10 9 TU/mL) that was fixed at 7 days post-virus (DPV) and co-labeled with antibodies to BrdU (red) and Ki-67 (green). Phalloidin labeling (grayscale) is shown to aid in visualizing the borders of the sensory epithelium. White dashed lines demarcate the borders of the sensory epithelium. Scale bar, 100 µm. Insets show high-resolution views of nuclei in the sensory epithelium. Arrows indicate nuclei that labeled with antibodies to BrdU but not Ki67. Scale bar for insets, 5 µm. ( B ) Graph shows the mean number of BrdU-labeled nuclei (green data points) and Ki-67-labeled nuclei (red data points) per sensory epithelium at 5, 7, and 10 DPV. ( C ) Graph shows quantification of the percentage of the BrdU-positive population that did not label with Ki67 antibodies (green data points) and the percentage of the Ki-67-positive population that did not label with BrdU antibodies (red data points). ( D ) Confocal image of an adult mouse utricle infected with Ad.MT58A (1×10 9 TU/mL) that was fixed at 7 DPV and co-labeled with antibodies to PH3-Ser10 (white) and Ki-67 (red). ( E ) Confocal images of a utricle from an embryonic day 17.5 (E17.5) mouse that was fixed in vivo and co-labeled with antibodies to PH3-Ser10 (white) and Ki-67 (red). Phalloidin labeling (grayscale) is shown to aid in visualizing the borders of the sensory epithelium. White dashed lines demarcate the borders of the sensory epithelium, and arrows in D and E indicate PH3-Ser10/Ki-67 co-labeled nuclei. Scale bar for D–E, 100 µm. ( F ) Graph shows quantification of the percentage of the Ki-67-positive populations that labeled with antibodies to PH3-Ser10. The difference in the percentage of PH3-Ser10-positive/Ki-67-positive nuclei did not reach statistical significance (p > 0.05; Student's t-test). The numbers above the gray bars indicate the mean number of PH3-Ser10-labeled nuclei per sensory epithelium. There were significantly more PH3-Ser10-positive nuclei in E17.5 utricles (p

    Article Snippet: Immunocytochemistry The following antibodies were used: rabbit anti-myosin VIIA (1∶200; Proteus Biosciences, Ramona, CA; # 25-6790) and mouse anti-myosin VIIA (1∶100; Developmental Studies Hybridoma Bank, Iowa City, Iowa; # MYO7A 138-1) to label hair cell soma; mouse anti-BrdU (1∶50; BD Biosciences; # 347580) to label cells that had incorporated BrdU during S-phase; rabbit anti-Ki67 (1∶200; Thermofisher Scientific, Kalamazoo, MI; # RM-9106-S0) to label cells in the active G1, S, G2, and M phases of the cell cycle; mouse anti-phospho-histone H3 (Ser10) (PH3-Ser10; 1∶200, Cell Signaling Technology, Danvers, MA; # 9706) to label cells in M phase; rabbit anti-Oct3/4 (1∶200; Santa Cruz Biotechnology, Santa Cruz, California; # SC-5279); mouse anti-Klf4 (1∶200; Abcam, Cambridge MA; # AB75486); rabbit anti-Sox2 (1∶200; Millipore, Billerica, MA; # AB5603); mouse anti-c-Myc (1∶200; Santa Cruz Biotechnology; # SC-40); mouse anti-HA (1∶200; Abcam; # AB18181); and rabbit anti-activated-caspase 3 to label cells undergoing apoptosis (1∶200; Abcam; # AB3623).

    Techniques: Infection, Labeling, In Vivo

    During normal development, the PDGFR-α-expressing oligodendrocyte progenitor population declines as the number of MBP-expressing mature oligodendrocytes increases. Cortical sections taken from wild-type animals at P0–P60 were stained for the oligodendrocyte progenitor marker PDGFR-α ( a–f ) and the mature oligodendrocyte marker MBP ( g–l ) using immunohistochemistry. Higher-magnification images of sections taken over the period from P0 to P60 stained with PDGFR-α, MBP and the proliferation marker Ki-67 demonstrate that proliferating oligodendrocyte progenitors diminish over time as myelination occurs, though proliferative PDGFR-α-expressing progenitors persist at least through 2 months of age ( o–t , arrows). Scale bars = 500 μm ( a, g ) and 50 μm ( a , inset, o ). m, n Western blot analysis of cortical samples taken from animals ranging in age from P0 to P60 was used to characterize PDGFR-α (180 kDa; m ) and MBP (14 kDa; n ) expression.

    Journal: Developmental Neuroscience

    Article Title: Age-Related Changes in the Oligodendrocyte Progenitor Pool Influence Brain Remodeling after Injury

    doi: 10.1159/000322081

    Figure Lengend Snippet: During normal development, the PDGFR-α-expressing oligodendrocyte progenitor population declines as the number of MBP-expressing mature oligodendrocytes increases. Cortical sections taken from wild-type animals at P0–P60 were stained for the oligodendrocyte progenitor marker PDGFR-α ( a–f ) and the mature oligodendrocyte marker MBP ( g–l ) using immunohistochemistry. Higher-magnification images of sections taken over the period from P0 to P60 stained with PDGFR-α, MBP and the proliferation marker Ki-67 demonstrate that proliferating oligodendrocyte progenitors diminish over time as myelination occurs, though proliferative PDGFR-α-expressing progenitors persist at least through 2 months of age ( o–t , arrows). Scale bars = 500 μm ( a, g ) and 50 μm ( a , inset, o ). m, n Western blot analysis of cortical samples taken from animals ranging in age from P0 to P60 was used to characterize PDGFR-α (180 kDa; m ) and MBP (14 kDa; n ) expression.

    Article Snippet: Primary antibodies used were mouse anti-myelin basic protein (anti-MBP; 1:500; Covance, Princeton, N.J., USA), rat anti-platelet-derived growth factor receptor-α (anti-PDGFR-α; 1:250; BD Pharmingen, Pasig City, Republic of the Philippines) and rabbit anti-Ki-67 (1:200; Thermo Scientific).

    Techniques: Expressing, Staining, Marker, Immunohistochemistry, Western Blot

    Dividing progenitors from nestin-HSV-TK transgenic mice are depleted when treated from P7 to P14. The pan-dividing cell marker Ki-67 was used in conjunction with PDGFR-α and MBP, and only in the animals treated from P7 to P14 were Ki-67-expressing dividing cells depleted ( a–h ). In animals treated from P14 to P28, there were fewer Ki-67-expressing cells in both the control and treated groups, with no differences noted between the two ( i–p ). Gan = Ganciclovir. Scale bar ( a ) = 100 μm.

    Journal: Developmental Neuroscience

    Article Title: Age-Related Changes in the Oligodendrocyte Progenitor Pool Influence Brain Remodeling after Injury

    doi: 10.1159/000322081

    Figure Lengend Snippet: Dividing progenitors from nestin-HSV-TK transgenic mice are depleted when treated from P7 to P14. The pan-dividing cell marker Ki-67 was used in conjunction with PDGFR-α and MBP, and only in the animals treated from P7 to P14 were Ki-67-expressing dividing cells depleted ( a–h ). In animals treated from P14 to P28, there were fewer Ki-67-expressing cells in both the control and treated groups, with no differences noted between the two ( i–p ). Gan = Ganciclovir. Scale bar ( a ) = 100 μm.

    Article Snippet: Primary antibodies used were mouse anti-myelin basic protein (anti-MBP; 1:500; Covance, Princeton, N.J., USA), rat anti-platelet-derived growth factor receptor-α (anti-PDGFR-α; 1:250; BD Pharmingen, Pasig City, Republic of the Philippines) and rabbit anti-Ki-67 (1:200; Thermo Scientific).

    Techniques: Transgenic Assay, Mouse Assay, Marker, Expressing

    Nicotine reduces tubular epithelial cell apoptosis and subsequent proliferation. Immunostaining of cleaved caspase-3 (A, B) and Ki67 (D, E) were performed one day and 3 days after I/R, respectively. Panel A represents TEC apoptosis in saline-treated animals (magnification ×40) and panel B represents TEC apoptosis in nicotine pre-treated animals (magnification ×40). Graph C compares the quantification of apoptotic TECs in saline-treated (black bars) or nicotine-treated animals (white bars), either after sham operation (n = 6 in each group) or renal I/R (n = 7 in saline-treated group and n = 10 in nicotine pretreated group). Panels D and E compare Ki-67+ cells between saline-treated animals and nicotine-treated animals (magnification ×40). Graph F compares the quantification of proliferation 3 days after reperfusion (n = 5 in each group). Data are expressed as mean ± SEM, (*) p≤0.05.

    Journal: PLoS ONE

    Article Title: Nicotine Protects Kidney from Renal Ischemia/Reperfusion Injury through the Cholinergic Anti-Inflammatory Pathway

    doi: 10.1371/journal.pone.0000469

    Figure Lengend Snippet: Nicotine reduces tubular epithelial cell apoptosis and subsequent proliferation. Immunostaining of cleaved caspase-3 (A, B) and Ki67 (D, E) were performed one day and 3 days after I/R, respectively. Panel A represents TEC apoptosis in saline-treated animals (magnification ×40) and panel B represents TEC apoptosis in nicotine pre-treated animals (magnification ×40). Graph C compares the quantification of apoptotic TECs in saline-treated (black bars) or nicotine-treated animals (white bars), either after sham operation (n = 6 in each group) or renal I/R (n = 7 in saline-treated group and n = 10 in nicotine pretreated group). Panels D and E compare Ki-67+ cells between saline-treated animals and nicotine-treated animals (magnification ×40). Graph F compares the quantification of proliferation 3 days after reperfusion (n = 5 in each group). Data are expressed as mean ± SEM, (*) p≤0.05.

    Article Snippet: For apoptose, sections were incubated overnight (4°C) with Cleaved Caspase-3 Antibody (Asp175; Cell Signaling) and for proliferation, sections were incubated overnight (4°C) with Rabbit Monoclonal anti-Ki67 (RM-9106; Lab Vision).

    Techniques: Immunostaining

    Epithelial proliferation after Nippostrongylus brasiliensis ( N.b. ) infection. ( a ) Representative photographs of lung tissues from naive or infected WT C57BL/6 mice at various time-points following subcutaneous infection of 650 L 3 N.b. . Arrows point to focal areas of hemorrhagic injury. All images were taken at 20× with Dino-Lite Pro Digital Microscope via Dino Capture 2.0 software. ( b ) Quantifications of Ym1 + , Ym1 + Ki-67 + , and Ki-67 + during N.b. infection. For each time-point, 5 randomly selected fields under 20× were counted. ( c ) Kinetic analysis of Ym1 (Cy2) and Ki-67 (Cy3) immunostaining within FFPE distal lung tissues of naïve or N.b. -infected mice at the time-points indicated. 200× magnification. ( d, e ) Images of ( d ) d3 and ( e ) d7 post-infection lung tissues at 400×. Yellow arrowheads indicate Ki-67 + cells and red arrowheads indicate Ki67 + /Ym1 + co-staining, respectively. DAPI counterstain is shown in blue. ( f ) Pseudo-color flow plots showing distal lung total live CD45 neg cells gated for EpCAM + cells that were BrdU + at the indicated time-points following N.b. infection. ( g ) Quantification of cells gated as in f isolated from WT C57BL/6 mice. ( h ) Comparison of cells gated as in f from WT, IL-4Rα −/− and RAGγc −/− strains at indicated time points. N=4 mice/group. Mean ±SEM are shown. *p

    Journal: Mucosal immunology

    Article Title: Macrophages promote epithelial proliferation following infectious and non-infectious lung injury through a Trefoil factor 2-dependent mechanism

    doi: 10.1038/s41385-018-0096-2

    Figure Lengend Snippet: Epithelial proliferation after Nippostrongylus brasiliensis ( N.b. ) infection. ( a ) Representative photographs of lung tissues from naive or infected WT C57BL/6 mice at various time-points following subcutaneous infection of 650 L 3 N.b. . Arrows point to focal areas of hemorrhagic injury. All images were taken at 20× with Dino-Lite Pro Digital Microscope via Dino Capture 2.0 software. ( b ) Quantifications of Ym1 + , Ym1 + Ki-67 + , and Ki-67 + during N.b. infection. For each time-point, 5 randomly selected fields under 20× were counted. ( c ) Kinetic analysis of Ym1 (Cy2) and Ki-67 (Cy3) immunostaining within FFPE distal lung tissues of naïve or N.b. -infected mice at the time-points indicated. 200× magnification. ( d, e ) Images of ( d ) d3 and ( e ) d7 post-infection lung tissues at 400×. Yellow arrowheads indicate Ki-67 + cells and red arrowheads indicate Ki67 + /Ym1 + co-staining, respectively. DAPI counterstain is shown in blue. ( f ) Pseudo-color flow plots showing distal lung total live CD45 neg cells gated for EpCAM + cells that were BrdU + at the indicated time-points following N.b. infection. ( g ) Quantification of cells gated as in f isolated from WT C57BL/6 mice. ( h ) Comparison of cells gated as in f from WT, IL-4Rα −/− and RAGγc −/− strains at indicated time points. N=4 mice/group. Mean ±SEM are shown. *p

    Article Snippet: Polyclonal goat anti-mouse Ym1 (R & D Systems,) and rabbit anti-mouse Ki67 (AbCam, Cambridge, MA), with secondary Alexa Fluor 488donkey anti-goat and Cy3-donkey anti-rabbit antibody (both from Jackson ImmunoResearch, West Grove, PA) respectively, were used in co-staining.

    Techniques: Infection, Mouse Assay, Microscopy, Software, Immunostaining, Formalin-fixed Paraffin-Embedded, Staining, Flow Cytometry, Isolation

    Immunohistochemical detection of Ki67 in Winnie distal colonic mucosa. A: Immunostaining of Ki67 in the distal colon of untreated C57BL6 mice. Image representative of Ki67 localisation in the distal colon of the four C57BL6 mice examined; B: Distal colonic Ki67 localisation representative of six C57BL6 mice exposed to three cycles of 1% dextran sulphate sodium (DSS); C: Distal colon of Winnie mouse without exposure to three cycles of DSS. Ki67-labelling in the epithelium is visible apically approximately half the crypt length; D: Ki67 immunolabelling of the Winnie distal colon exposed to three cycles of DSS. Crypt base proliferative zone extends approximately two-thirds of the crypt length. Submucosal gland (arrowhead) displays few positive nuclei. Scale bar represents a distance of 50 μm.

    Journal: World Journal of Gastroenterology

    Article Title: Characterisation of colonic dysplasia-like epithelial atypia in murine colitis

    doi: 10.3748/wjg.v22.i37.8334

    Figure Lengend Snippet: Immunohistochemical detection of Ki67 in Winnie distal colonic mucosa. A: Immunostaining of Ki67 in the distal colon of untreated C57BL6 mice. Image representative of Ki67 localisation in the distal colon of the four C57BL6 mice examined; B: Distal colonic Ki67 localisation representative of six C57BL6 mice exposed to three cycles of 1% dextran sulphate sodium (DSS); C: Distal colon of Winnie mouse without exposure to three cycles of DSS. Ki67-labelling in the epithelium is visible apically approximately half the crypt length; D: Ki67 immunolabelling of the Winnie distal colon exposed to three cycles of DSS. Crypt base proliferative zone extends approximately two-thirds of the crypt length. Submucosal gland (arrowhead) displays few positive nuclei. Scale bar represents a distance of 50 μm.

    Article Snippet: Slides were incubated with either anti-human β-catenin (clone E247; Abcam, Cambridge, United Kingdom), at a 1:500 dilution, rabbit anti-human Ki67 (clone SP6; Abcam) or rabbit anti-mouse Cxcl5 (Bioss Inc., Woburn, MA, United States) at a dilution of 1:100 was incubated with the slides for 1 h. Excess primary antibody was removed with 3 × 2 min washes with TBS prior to application of HRP-conjugated anti-rabbit secondary antibody (Biocare Medical) for 30 min.

    Techniques: Immunohistochemistry, Immunostaining, Mouse Assay

    BEZ235 and LY3203414 decrease cellular proliferation and increase differentiation in Apc and Pik3ca mutant colon cancer cells. AP spheroids grown in Matrigel on glass coverslips were treated with NVP-BYL719, BEZ235, LY3203414 or control for 24 hours. A decrease in the percent of cells staining for Ki67 was observed in those spheres treated with BEZ235 (p = 0.037) or LY3203414 (p = 0.01), but not NVP-BYL719 (p = 0.74; A). No significant differences were observed in staining for cleaved caspase 3 (B). An increase in staining for keratin 20, a marker of cellular differentiation was observed with BEZ235 and LY3023414 compared to NVP-BEZ719 and control (C).

    Journal: Molecular cancer research : MCR

    Article Title: Dual PI3K/mTOR Inhibition in Colorectal Cancers with APC and PIK3CA Mutations

    doi: 10.1158/1541-7786.MCR-16-0256

    Figure Lengend Snippet: BEZ235 and LY3203414 decrease cellular proliferation and increase differentiation in Apc and Pik3ca mutant colon cancer cells. AP spheroids grown in Matrigel on glass coverslips were treated with NVP-BYL719, BEZ235, LY3203414 or control for 24 hours. A decrease in the percent of cells staining for Ki67 was observed in those spheres treated with BEZ235 (p = 0.037) or LY3203414 (p = 0.01), but not NVP-BYL719 (p = 0.74; A). No significant differences were observed in staining for cleaved caspase 3 (B). An increase in staining for keratin 20, a marker of cellular differentiation was observed with BEZ235 and LY3023414 compared to NVP-BEZ719 and control (C).

    Article Snippet: The primary antibodies included: Ki67 (#12202, 1:400, Cell Signaling Technology, Danvers, MA), phospho ERK 1/2 (Thr202/Tyr204, #4370, 1:400, Cell Signaling Technology), CTNNB1 (#8480, 1:200, Cell Signaling Technology), pAKT (Ser473, #4060, 1:100 Cell Signaling Technology), and phospho ribosomal protein S6 (RPS6) (Ser235/236, #4858, 1:50, Cell Signaling Technology).

    Techniques: Mutagenesis, Staining, Marker, Cell Differentiation

    Moderately differentiated adenocarcinomas form within the colon of AP mice (A, B) and can be cultured using 3-dimensional techniques (C). These cancers can be grown as spheroids that form hollow spheres (D) and can even develop crypt-like structures (E-G). In spheroid culture, AP spheres retain many characteristics of AP cancers, including a similar morphology, nuclear localization of CTNNB1 (β-catenin), phosphorylation of RPS6, and expression of Ki67 (H). Proliferation of these spheres can be monitored over time with serial imaging (I). Size bars: A, 1 cm; D and E, 500 µm; H, 200 µm; I, 500 µm. B is a 5x enlargement of the area outlined in A; F is a 5x enlargement of E; and G is a 2x enlargement of F.

    Journal: Molecular cancer research : MCR

    Article Title: Dual PI3K/mTOR Inhibition in Colorectal Cancers with APC and PIK3CA Mutations

    doi: 10.1158/1541-7786.MCR-16-0256

    Figure Lengend Snippet: Moderately differentiated adenocarcinomas form within the colon of AP mice (A, B) and can be cultured using 3-dimensional techniques (C). These cancers can be grown as spheroids that form hollow spheres (D) and can even develop crypt-like structures (E-G). In spheroid culture, AP spheres retain many characteristics of AP cancers, including a similar morphology, nuclear localization of CTNNB1 (β-catenin), phosphorylation of RPS6, and expression of Ki67 (H). Proliferation of these spheres can be monitored over time with serial imaging (I). Size bars: A, 1 cm; D and E, 500 µm; H, 200 µm; I, 500 µm. B is a 5x enlargement of the area outlined in A; F is a 5x enlargement of E; and G is a 2x enlargement of F.

    Article Snippet: The primary antibodies included: Ki67 (#12202, 1:400, Cell Signaling Technology, Danvers, MA), phospho ERK 1/2 (Thr202/Tyr204, #4370, 1:400, Cell Signaling Technology), CTNNB1 (#8480, 1:200, Cell Signaling Technology), pAKT (Ser473, #4060, 1:100 Cell Signaling Technology), and phospho ribosomal protein S6 (RPS6) (Ser235/236, #4858, 1:50, Cell Signaling Technology).

    Techniques: Mouse Assay, Cell Culture, Expressing, Imaging