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  • 99
    Thermo Fisher oligo dt
    Runx2 contributes to regulation of the TGFβ mediated activation of PTHrP in cancer cells MDA-MB-231 cells were transfected with Runx2 siRNA <t>oligo</t> (#2) or control oligos (NS) for 36 h followed by TGFβ (5nM) treatment for another 24 h. ( a ): The knock down levels of Runx2 was confirmed by western blot analysis. Cells were harvested for total <t>RNA</t> to detect IHH ( b ) or PTHrP ( c ) levels by qRT-PCR analysis. ( c: right panel ): PTHrP levels in MDA-MB-231 cells treated with cyclopamine (Cyclo: 5µM) followed by TGFβ induction (5nM) for 24 h. Over expression of Runx2 increased TGFβ responsiveness to PTHrP . ( d ): PTHrP mRNA levels in MDA-MB-231 cells transduced with control or Runx2 adenovirus followed by TGFβ treatment.
    Oligo Dt, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 27227 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore poly d lysine
    Matrix switching during migration affects motility disproportionally to the nature of the matrix (a) Using Star MAts HEp3 cell migration was analyzed on uniform substrates of collagen (CN), <t>fibronectin</t> (FN), and laminin (LN). The migration rates are displayed in (b) as from CN onto CN, from FN onto FN, and so on. (c) Using Block MAts, two adjacent matrixes were created as described in Methods. Cells were seeded onto one matrix, cultured to confluence, and subsequently allowed to migrate onto the adjacent matrix. Images taken at 0, 8, and 16 hours document migration and the morphological changes visible when HEp3 cells switch from CN onto adjacent <t>poly-D-lysine</t> (PDL), FN, and LN, or vice versa. (b) The quantitation of migration rates across these interfaces revealed significant matrix-specific effects and unexpected migration deficiencies that could only be contributed to matrix switching and not the matrix itself (CN vs. FN). Images were taken on the Axiovert 135 microscope. Microscope and boxplot details are described in Methods. Scale bars, 50 μm.
    Poly D Lysine, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 15561 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore actinomycin d
    Stabilization of LSAT mRNA by A23187 does not involve new transcription. HeLa cells were transfected with pLSGC or pLSAT plasmids, treated with 5 μM <t>actinomycin</t> D for 30 min to block new transcription, then treated with 10 μM A23187 for the times indicated (lanes 1–6). In lane 7, transcription was blocked for 1 h with actinomycin D without A23187 treatment. Total cytoplasmic RNA was purified, equal amounts resolved by Northern analysis and hybridized to probes for S-antigen or GAPDH.
    Actinomycin D, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 12946 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher pentr d topo
    The Entry/Gateway ® cloning procedures for generating epitope-tagged fusion proteins using the P-element destination vectors . The procedures of Entry/Gateway ® cloning are illustrated in the diagram as two major steps. (1) In the first step, a fragment encoding the open reading frame (ORF) is inserted into an entry vector to generate entry clones. Two entry vectors were described in this study: ( i ) the pGWS which uses a TA-based method; ( ii ) <t>pENTR/D-TOPO</t> (Invitrogen) which uses a TOPO-based method. (2) In the second step, the ORF in the entry clone is recombined into one of the P-DEST vectors.
    Pentr D Topo, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 10828 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore cytochalasin b
    Increased levels of DNA damage in CSB mutant cells after a prolongued 5-azadC treatment (A–D) AA8 and UV61 cells were treated for 24 h with 5-azadC (2μM) washed and allowed to repair for 24h. Then were fixed and prepared for immunological staining of γ-H2AX (A-B), 53BP1 (C) and RAD51 (D). (E) In order to monitor residual DNA damage after a 24h treatment with 5-azadC, we performed micronucleus assay. Cells were treated with increasing doses of 5-azadC, washed and allowed to repair in <t>cytochalasin</t> B containing media for 18h. Cells were then fixed and scored for the number of micronuclei in binucleated cells. The means and SEM of three independent experiments are shown. Data were statistically analysed using Student's t -test. Data were considered statistically different when P
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    99
    Millipore 2 deoxy d glucose
    p53 promotes oxidative phosphorylation (A) The fluorescence lifetime histogram on the phasor plot shows the distribution of lifetimes of autofluorescent NADH. A color spectrum indicates the bound fraction of NADH. (B) Fluorescence intensity images of GFP of an EGFP cell and p53-GFP cell (left panels). Normalized fluorescence intensity images of NADH (middle panels) and pseudo-colored FLIM images (right panels) of H1299 lung cancer cells transfected with EGFP and p53-GFP. Pseudo-coloring corresponds to the phasor position according to the color spectrum in Figure 1A. Scale bar = 10μm. (C) Bar graphs quantifying the bound fraction of NADH in the nucleus (left) and cytoplasm (right). (D) Percent change in bound fraction of NADH in the nucleus (left) and cytoplasm (right) after treatment with DMSO, rotenone and antimycin A (R+AA), or <t>2-deoxy-D-glucose</t> and dichloroacetate (DG+DCA). * p
    2 Deoxy D Glucose, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1480 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher pentr d topo vector
    p53 promotes oxidative phosphorylation (A) The fluorescence lifetime histogram on the phasor plot shows the distribution of lifetimes of autofluorescent NADH. A color spectrum indicates the bound fraction of NADH. (B) Fluorescence intensity images of GFP of an EGFP cell and p53-GFP cell (left panels). Normalized fluorescence intensity images of NADH (middle panels) and pseudo-colored FLIM images (right panels) of H1299 lung cancer cells transfected with EGFP and p53-GFP. Pseudo-coloring corresponds to the phasor position according to the color spectrum in Figure 1A. Scale bar = 10μm. (C) Bar graphs quantifying the bound fraction of NADH in the nucleus (left) and cytoplasm (right). (D) Percent change in bound fraction of NADH in the nucleus (left) and cytoplasm (right) after treatment with DMSO, rotenone and antimycin A (R+AA), or <t>2-deoxy-D-glucose</t> and dichloroacetate (DG+DCA). * p
    Pentr D Topo Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 9046 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore nmda
    Dendritic release requires L-type Ca 2+ channels. A , Dendritic release responses to <t>AMPA</t> or <t>NMDA</t> in the presence and absence of IEM1460 (100 µM) or nimodipine (3 µM). n ≥ 5. ****p
    Nmda, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1815 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    GE Healthcare 2 d quant kit
    Comparative proteomic analysis of vehicle- and endorepellin-treated human endothelial cells . Representative <t>2-D</t> protein profiles from vehicle- and endorepellin-treated endothelial cells are depicted with protein separation according to isoelectric point (pI, x-axis) and molecular weight (kDa, y-axis). Encircled protein spots correspond to the five proteins differentially expressed in response to endorepellin treatment. Compare calreticulin (1), β-actin (2), chaperonin/Hsp60 (3), vimentin (4) and prolyl 4-hydorxylase, β subunit (5), spot intensities in the two sets of samples. The spots under the bracket represent serum albumin by LC-MS/MS.
    2 D Quant Kit, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 4938 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Becton Dickinson 7 amino actinomycin d
    Nocodazole Does Not Inhibit rAAV2 Transduction ( a ) Microtubule and Golgi morphology. HeLa cells were preincubated in 1µM nocodazole for 4 hours and then fixed to observe the microtubule morphology (anti-beta-tubulin, Cy2, green) and Golgi localization (anti-p230, Cy3, red). Nuclei are in blue (DAPI). Scale bar = 25µm. ( b ) Percent GFP+ cells, normalized to no inhibitor control, as a function of nocodazole concentrations. After 4 hours preincubation with the nocodazole concentrations indicated, HeLa cells were transduced with rAAV2-GFP at 10 4 gcp/cell. Twenty-four hours after transduction, the cells were harvested and stained with <t>7-amino-actinomycin</t> D (7AAD) prior to FACS analysis for GFP. The GFP+ percentages were determined on the 7AAD− subset of a coherent population first defined by FSC/SSC. The %GFP populations were then normalized to a no inhibitor control represented by mean ± SD. ( c ) Normalized viability as a function of nocodazole concentrations. Viability was defined as FSC/SSC coherent, 7AAD− population, which was then normalized to a no inhibitor control.
    7 Amino Actinomycin D, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 3958 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    IDEX investissements d avenir
    Nocodazole Does Not Inhibit rAAV2 Transduction ( a ) Microtubule and Golgi morphology. HeLa cells were preincubated in 1µM nocodazole for 4 hours and then fixed to observe the microtubule morphology (anti-beta-tubulin, Cy2, green) and Golgi localization (anti-p230, Cy3, red). Nuclei are in blue (DAPI). Scale bar = 25µm. ( b ) Percent GFP+ cells, normalized to no inhibitor control, as a function of nocodazole concentrations. After 4 hours preincubation with the nocodazole concentrations indicated, HeLa cells were transduced with rAAV2-GFP at 10 4 gcp/cell. Twenty-four hours after transduction, the cells were harvested and stained with <t>7-amino-actinomycin</t> D (7AAD) prior to FACS analysis for GFP. The GFP+ percentages were determined on the 7AAD− subset of a coherent population first defined by FSC/SSC. The %GFP populations were then normalized to a no inhibitor control represented by mean ± SD. ( c ) Normalized viability as a function of nocodazole concentrations. Viability was defined as FSC/SSC coherent, 7AAD− population, which was then normalized to a no inhibitor control.
    Investissements D Avenir, supplied by IDEX, used in various techniques. Bioz Stars score: 94/100, based on 776 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Caliper Life Sciences d luciferin
    Nocodazole Does Not Inhibit rAAV2 Transduction ( a ) Microtubule and Golgi morphology. HeLa cells were preincubated in 1µM nocodazole for 4 hours and then fixed to observe the microtubule morphology (anti-beta-tubulin, Cy2, green) and Golgi localization (anti-p230, Cy3, red). Nuclei are in blue (DAPI). Scale bar = 25µm. ( b ) Percent GFP+ cells, normalized to no inhibitor control, as a function of nocodazole concentrations. After 4 hours preincubation with the nocodazole concentrations indicated, HeLa cells were transduced with rAAV2-GFP at 10 4 gcp/cell. Twenty-four hours after transduction, the cells were harvested and stained with <t>7-amino-actinomycin</t> D (7AAD) prior to FACS analysis for GFP. The GFP+ percentages were determined on the 7AAD− subset of a coherent population first defined by FSC/SSC. The %GFP populations were then normalized to a no inhibitor control represented by mean ± SD. ( c ) Normalized viability as a function of nocodazole concentrations. Viability was defined as FSC/SSC coherent, 7AAD− population, which was then normalized to a no inhibitor control.
    D Luciferin, supplied by Caliper Life Sciences, used in various techniques. Bioz Stars score: 94/100, based on 3742 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Promega d luciferin
    Nocodazole Does Not Inhibit rAAV2 Transduction ( a ) Microtubule and Golgi morphology. HeLa cells were preincubated in 1µM nocodazole for 4 hours and then fixed to observe the microtubule morphology (anti-beta-tubulin, Cy2, green) and Golgi localization (anti-p230, Cy3, red). Nuclei are in blue (DAPI). Scale bar = 25µm. ( b ) Percent GFP+ cells, normalized to no inhibitor control, as a function of nocodazole concentrations. After 4 hours preincubation with the nocodazole concentrations indicated, HeLa cells were transduced with rAAV2-GFP at 10 4 gcp/cell. Twenty-four hours after transduction, the cells were harvested and stained with <t>7-amino-actinomycin</t> D (7AAD) prior to FACS analysis for GFP. The GFP+ percentages were determined on the 7AAD− subset of a coherent population first defined by FSC/SSC. The %GFP populations were then normalized to a no inhibitor control represented by mean ± SD. ( c ) Normalized viability as a function of nocodazole concentrations. Viability was defined as FSC/SSC coherent, 7AAD− population, which was then normalized to a no inhibitor control.
    D Luciferin, supplied by Promega, used in various techniques. Bioz Stars score: 92/100, based on 3717 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher 7 aad
    Mechanisms for KLRG1 + CD8 T cells suppressing tumors. ( a ) KLRG1 + CD8 T cells or KLRG1 − CD8 T cells were co-cultured with B16-GFP cells (green) at the E:T ratio of 5:1, and the killing process was captured by PE spinning disk live cell confocal microscope with a 60 × oil immersion lens. ( b ) KLRG1 + CD8 T cells or KLRG1 − CD8 T cells were co-cultured with B16-GFP cells at the E:T ratio of 5:1 for 24 hours. Then target cells were collected and stained with <t>7-AAD.</t> Percentages of 7-AAD positive populations indicated the killing rates. ( c ) KLRG1 + CD8 T cells or KLRG1 − CD8 T cells were co-cultured with EL4 cells at the E:T ratio of 20:1 for 12 hours. Then target cells were collected and stained with 7-AAD. Percentages of 7-AAD positive populations indicated the killing rates. ( d ) KLRG1 + CD8 T cells and KLRG1 − CD8 T cells were co-cultured with EL4 cells at the E:T ratio of 5:1 and 20:1 for 24 h with or without 50ug/mL anti-FasL, 50ug/mL anti-TRAIL, and 50 μM Granzyme B inhibitor Z-AAD-CMK. Cytotoxicity against target cells was evaluated and shown. ( e ) In an in vitro matrigel invasion experiment, KLRG1 + CD8 T cells or KLRG1 − CD8 T cells were sorted and inoculated on the upper layer. After 24 hours, penetrated cells on the lower layer were collected and calculated. ( f – h ) Real-time PCR ( f , g ) was carried out to examine the gene expression of heparanase and p53, which were also confirmed by RNA-seq analysis. ( h ) In vitro experiments were performed in triplicates for three times.
    7 Aad, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3718 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore isopropyl β d thiogalactopyranoside
    Expression of HC29 protein and Western blotting. Gels were stained with Coomassie blue, and 15 µL samples were loaded per lane. Lane PM: Standard protein molecular weight marker, Lane 1: Extracts of Escherichia ( E. ) coli (BL21 strain) transformed with pET-28a empty expression vector before induction with <t>isopropyl-β-D-thiogalactopyranoside</t> (IPTG, negative control), Lane 2: Extracts of E. coli (BL21 strain) transformed with pET-28a empty expression vector after 5 h of induction with IPTG (negative control), Lane 3: Extracts of E. coli (BL21 strain) transformed with pET-28a/HC29 before induction with IPTG, Lanes 4~8: Extracts of E. coli (BL21 strain) transformed with pET-28a/HC29 after 1~5 h of induction with IPTG, Lane 9: Purified recombinant HC29 protein supernatants, Lane 10: HC29 natural protein in soluble proteins of Haemonchus contortus was identified using rat sera as a primary antibody.
    Isopropyl β D Thiogalactopyranoside, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3787 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore deoxycholic acid
    <t>Deoxycholic</t> acid does not disrupt endothelial tight junctions via known signal transduction pathways. Confluent monolayers of brain microvessel endothelial cells were treated for 24 hours with vehicle or deoxycholic acid in either the presence or absence
    Deoxycholic Acid, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2295 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore dextrose
    <t>Deoxycholic</t> acid does not disrupt endothelial tight junctions via known signal transduction pathways. Confluent monolayers of brain microvessel endothelial cells were treated for 24 hours with vehicle or deoxycholic acid in either the presence or absence
    Dextrose, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 639 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Tocris d ap5
    CT-facilitated reversal of LTP is NMDA receptor-dependent. Stable LTP was induced with HFS ( white arrow ) in animals injected with the NMDA receptor antagonist <t>D-AP5</t> (100 nmol; black arrowhead ) 5 min before the administration of CT 105 (1 pmol) and 5 min
    D Ap5, supplied by Tocris, used in various techniques. Bioz Stars score: 93/100, based on 886 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore collagenase d
    Cetuximab modulates macrophage polarization in an AOM/DSS mouse model A. Establishment of the AOM/DSS mouse model. AOM was injected intraperitoneally at 12.5 mg/kg body weight. After one week, mice were given drinking water containing 2.5% DSS for 5 days followed by 16 days of regular drinking water. After two cycles of DSS treatment, cetuximab (1 mg/mouse, twice a week) was injected intraperitoneally for a month, and the mice were then sacrificed. B. Representative images of colon tumors in normal (right), AOM/DSS (2AD) (middle), and cetuximab-treated AOM/DSS mice (2AD+cetu) (left). C. Tumor quantification. Cetuximab treatment (2AD + cetu) reduced tumor numbers compared to 2AD mice. D. p-EGFR (Y1068), EGFR, Arg1, and iNOS protein levels in normal mice, 2AD and 2AD+cetu mouse tumors were detected by Western blot. All experiments were repeated three times. E. Representative photomicrographs of immunostaining for p-EGFR (Y1068), PCNA, F4/80, Arg1, and iNOS. Scale bars: 25 μm. F. M1 marker (IL-12, iNOS) and M2 marker (IL-4, IL-10, Arg1) mRNA levels in normal mouse colon tissues, 2AD and 2AD+cetu mouse tumor tissues were evaluated by q-PCR. G. Percentages of CD11b + /F4/80 + and F4/80 + /CD206 + cells in normal, 2AD, and 2AD+cetu mice colon tissues were detected by flow cytometry. Colon tissues were cut into small pieces (1-2 mm) and incubated with <t>collagenase</t> D (1- mg/mL), dispase II (1 mg/mL), and DNase I (100 μg/mL) for 30-45 min in a shaking incubator at 37°C, and single-cell suspensions were then incubated with antibodies. Bars represent means ± SD (n = 3) for each treatment. * p
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    Millipore d amphetamine sulfate
    Cetuximab modulates macrophage polarization in an AOM/DSS mouse model A. Establishment of the AOM/DSS mouse model. AOM was injected intraperitoneally at 12.5 mg/kg body weight. After one week, mice were given drinking water containing 2.5% DSS for 5 days followed by 16 days of regular drinking water. After two cycles of DSS treatment, cetuximab (1 mg/mouse, twice a week) was injected intraperitoneally for a month, and the mice were then sacrificed. B. Representative images of colon tumors in normal (right), AOM/DSS (2AD) (middle), and cetuximab-treated AOM/DSS mice (2AD+cetu) (left). C. Tumor quantification. Cetuximab treatment (2AD + cetu) reduced tumor numbers compared to 2AD mice. D. p-EGFR (Y1068), EGFR, Arg1, and iNOS protein levels in normal mice, 2AD and 2AD+cetu mouse tumors were detected by Western blot. All experiments were repeated three times. E. Representative photomicrographs of immunostaining for p-EGFR (Y1068), PCNA, F4/80, Arg1, and iNOS. Scale bars: 25 μm. F. M1 marker (IL-12, iNOS) and M2 marker (IL-4, IL-10, Arg1) mRNA levels in normal mouse colon tissues, 2AD and 2AD+cetu mouse tumor tissues were evaluated by q-PCR. G. Percentages of CD11b + /F4/80 + and F4/80 + /CD206 + cells in normal, 2AD, and 2AD+cetu mice colon tissues were detected by flow cytometry. Colon tissues were cut into small pieces (1-2 mm) and incubated with <t>collagenase</t> D (1- mg/mL), dispase II (1 mg/mL), and DNase I (100 μg/mL) for 30-45 min in a shaking incubator at 37°C, and single-cell suspensions were then incubated with antibodies. Bars represent means ± SD (n = 3) for each treatment. * p
    D Amphetamine Sulfate, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 479 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Runx2 contributes to regulation of the TGFβ mediated activation of PTHrP in cancer cells MDA-MB-231 cells were transfected with Runx2 siRNA oligo (#2) or control oligos (NS) for 36 h followed by TGFβ (5nM) treatment for another 24 h. ( a ): The knock down levels of Runx2 was confirmed by western blot analysis. Cells were harvested for total RNA to detect IHH ( b ) or PTHrP ( c ) levels by qRT-PCR analysis. ( c: right panel ): PTHrP levels in MDA-MB-231 cells treated with cyclopamine (Cyclo: 5µM) followed by TGFβ induction (5nM) for 24 h. Over expression of Runx2 increased TGFβ responsiveness to PTHrP . ( d ): PTHrP mRNA levels in MDA-MB-231 cells transduced with control or Runx2 adenovirus followed by TGFβ treatment.

    Journal: Cancer research

    Article Title: Runx2 Transcriptional Activation of Indian Hedgehog and a Downstream Bone Metastatic Pathway in Breast Cancer Cells

    doi: 10.1158/0008-5472.CAN-08-1078

    Figure Lengend Snippet: Runx2 contributes to regulation of the TGFβ mediated activation of PTHrP in cancer cells MDA-MB-231 cells were transfected with Runx2 siRNA oligo (#2) or control oligos (NS) for 36 h followed by TGFβ (5nM) treatment for another 24 h. ( a ): The knock down levels of Runx2 was confirmed by western blot analysis. Cells were harvested for total RNA to detect IHH ( b ) or PTHrP ( c ) levels by qRT-PCR analysis. ( c: right panel ): PTHrP levels in MDA-MB-231 cells treated with cyclopamine (Cyclo: 5µM) followed by TGFβ induction (5nM) for 24 h. Over expression of Runx2 increased TGFβ responsiveness to PTHrP . ( d ): PTHrP mRNA levels in MDA-MB-231 cells transduced with control or Runx2 adenovirus followed by TGFβ treatment.

    Article Snippet: Purified RNA was oligo dT primed and cDNA synthesized at 42°C with SuperScript II RNA polymerase (Invitrogen).

    Techniques: Activation Assay, Multiple Displacement Amplification, Transfection, Western Blot, Quantitative RT-PCR, Over Expression, Transduction

    Midkine expression was induced by FBS, growth factors and cytokines. A . LNCaP cells were cultured in serum-free DMEM and treated for 48 h with 10% FBS and the indicated agents: 10 ng/ml insulin, 10 ng/ml IGF-I, 10 ng/ml EGF, 10 ng/ml HGF, 10 ng/ml bFGF, 20 ng/ml T3, 10 nM R1881, 10 nM DHT, 10 ng/ml TNFα, 10 ng/ml IL-1β, 50 ng/ml IL-6, 50 ng/ml IL-17, and 33.3 μM RA. B . LNCaP cells were treated with different dosages (1 to 50 ng/ml) of TNFα for 48 h. C . LNCaP cells were also treated with 20 ng/ml TNFα for different time periods (8 to 48 h). 20 μl of each medium supernatant was subjected to Western blot analysis of midkine expression using rabbit anti-midkine antibodies, horseradish peroxidase-conjungated secondary antibodies and enhanced chemiluminescence reagents. D . Total RNA was extracted from LNCaP cells not treated or treated with 20 ng/ml TNFα, using RNeasy Mini Kit; cDNA was made from total RNA using Superscript™ First-Strand Synthesis System with oligo dT primers; real-time quantitative PCR was done in triplicates with Sybr-Green reagents; results were normalized to GAPDH levels as described in Methods; the data (mean ± standard deviation of three independent experiments) were presented as fold change of midkine mRNA compared to the LNCaP cells without treatment for 8 h, where fold = 2 ΔΔCt ; solid bar, TNFα treated; open bar, control; * P

    Journal: BMC Medical Genomics

    Article Title: Midkine is a NF-?B-inducible gene that supports prostate cancer cell survival

    doi: 10.1186/1755-8794-1-6

    Figure Lengend Snippet: Midkine expression was induced by FBS, growth factors and cytokines. A . LNCaP cells were cultured in serum-free DMEM and treated for 48 h with 10% FBS and the indicated agents: 10 ng/ml insulin, 10 ng/ml IGF-I, 10 ng/ml EGF, 10 ng/ml HGF, 10 ng/ml bFGF, 20 ng/ml T3, 10 nM R1881, 10 nM DHT, 10 ng/ml TNFα, 10 ng/ml IL-1β, 50 ng/ml IL-6, 50 ng/ml IL-17, and 33.3 μM RA. B . LNCaP cells were treated with different dosages (1 to 50 ng/ml) of TNFα for 48 h. C . LNCaP cells were also treated with 20 ng/ml TNFα for different time periods (8 to 48 h). 20 μl of each medium supernatant was subjected to Western blot analysis of midkine expression using rabbit anti-midkine antibodies, horseradish peroxidase-conjungated secondary antibodies and enhanced chemiluminescence reagents. D . Total RNA was extracted from LNCaP cells not treated or treated with 20 ng/ml TNFα, using RNeasy Mini Kit; cDNA was made from total RNA using Superscript™ First-Strand Synthesis System with oligo dT primers; real-time quantitative PCR was done in triplicates with Sybr-Green reagents; results were normalized to GAPDH levels as described in Methods; the data (mean ± standard deviation of three independent experiments) were presented as fold change of midkine mRNA compared to the LNCaP cells without treatment for 8 h, where fold = 2 ΔΔCt ; solid bar, TNFα treated; open bar, control; * P

    Article Snippet: Analysis of MDK mRNA expression by real-time quantitative RT-PCR Total RNA was extracted from LNCaP cells not treated or treated with 20 ng/ml TNFα, using RNeasy Mini Kit (QIAGEN, Valencia, CA) with on-membrane DNase I digestion to avoid genomic DNA contamination. cDNA was made from total RNA using Superscript™ First-Strand Synthesis System with oligo dT primers (Invitrogen, Carlsbad, CA).

    Techniques: Expressing, Cell Culture, Western Blot, Real-time Polymerase Chain Reaction, SYBR Green Assay, Standard Deviation

    E-cadherin plays a pivotal role in miR-BART9-mediated migration and invasion in NPC cells. (A) Protein level of E-cadherin was increased after introducing pcDNA6/His-CDH1, which contains CDH1 open reading frame without 3′-UTR. Transwell migration assay (B) and Matrigel invasion assay (C) for miR-BART9- or LacZ-expressing BM1 cells with or without ectopic expression of E-cadherin. (D) HK1-EBV cells were transfected with 10 nM siRNA negative control (si-Neg) or CDH1 siRNA (si-CDH1). Expression of E-cadherin was examined by Western blotting. GAPDH was used as a loading control. (E) Transwell migration assay (Upper) and Matrigel invasion assay (Middle) of HK1-EBV cells treated with an LNA-modified miR-BART9 antisense oligo (anti-BART9), scramble control (anti-Ctrl), or anti-BART9 plus E-cadherin siRNA (si-CHD1). Images of cells adhered to the lower surface of the filter insert from a representative experiment are shown. The numbers of migratory or invasive cells were quantified using image J and are expressed as the fold change relative to the appropriate cell line (bar graphs). The data are expressed as the means ± SEM from three independent experiments and two-tailed Student's t-tests were performed (*, P

    Journal: PLoS Pathogens

    Article Title: The Epstein-Barr Virus-Encoded MicroRNA MiR-BART9 Promotes Tumor Metastasis by Targeting E-Cadherin in Nasopharyngeal Carcinoma

    doi: 10.1371/journal.ppat.1003974

    Figure Lengend Snippet: E-cadherin plays a pivotal role in miR-BART9-mediated migration and invasion in NPC cells. (A) Protein level of E-cadherin was increased after introducing pcDNA6/His-CDH1, which contains CDH1 open reading frame without 3′-UTR. Transwell migration assay (B) and Matrigel invasion assay (C) for miR-BART9- or LacZ-expressing BM1 cells with or without ectopic expression of E-cadherin. (D) HK1-EBV cells were transfected with 10 nM siRNA negative control (si-Neg) or CDH1 siRNA (si-CDH1). Expression of E-cadherin was examined by Western blotting. GAPDH was used as a loading control. (E) Transwell migration assay (Upper) and Matrigel invasion assay (Middle) of HK1-EBV cells treated with an LNA-modified miR-BART9 antisense oligo (anti-BART9), scramble control (anti-Ctrl), or anti-BART9 plus E-cadherin siRNA (si-CHD1). Images of cells adhered to the lower surface of the filter insert from a representative experiment are shown. The numbers of migratory or invasive cells were quantified using image J and are expressed as the fold change relative to the appropriate cell line (bar graphs). The data are expressed as the means ± SEM from three independent experiments and two-tailed Student's t-tests were performed (*, P

    Article Snippet: For mRNA quantification, 1 µg of total RNA was employed as a template for reverse transcription using an oligo dT primer and Superscript III (Invitrogen).

    Techniques: Migration, Transwell Migration Assay, Invasion Assay, Expressing, Transfection, Negative Control, Western Blot, Modification, Two Tailed Test

    Depletion of endogenous miR-BART9 suppresses the migration and invasiveness of EBV-positive NPC cells. (A) LNA-modified anti-BART9 efficiently decreases the level of mature miR-BART9 in EBV-positive HK1-EBV and C666-1 cells. HK1-EBV and C666-1 cells were treated with a 12.5 nM concentration of an LNA-modified miR-BART9 antisense oligo (anti-BART9) or a scramble control (anti-Ctrl) for 48 hr. The expression level of miR-BART9 was determined via qPCR. (B) HK1-EBV cells were treated with anti-BART9 or anti-Ctrl for 24 hr and the plated for colony formation assays. Colony formation activity was determined via crystal violet staining after 11 days in culture. (C, D) Transwell migration assay (C) and Matrigel invasion assay (D) for HK1-EBV and C666-1 cells. Cells were treated with a 12.5 nM concentration of an LNA-modified miR-BART9 antisense oligo (anti-BART9) or a scramble control (anti-Ctrl) for 48 hr before the migration or invasion assay. Images of cells adhered to the lower surface of the filter insert from a representative experiment are shown. (Left panel). The numbers of migratory or invasive cells were quantified using image J and expressed as the fold change relative to the appropriate cell line (bar graphs). (E) Expression levels of LMP1, LMP2A and EBNA1 in HK1-EBV and C666-1 cells treated with a 12.5 nM concentration of an LNA-modified miR-BART9 antisense oligo (anti-BART9) or a scramble control (anti-Ctrl). Total RNA was collected 48 hr after transfection and mRNA levels were determined via qPCR. The data were normalized to cellular EEF1A1 levels and expressed as the fold change relative to the appropriate cell line. The bar graphs in (B), (C), (D), (E) show means ± SEM from three independent experiments and two-tailed Student's t-tests were performed (*, P

    Journal: PLoS Pathogens

    Article Title: The Epstein-Barr Virus-Encoded MicroRNA MiR-BART9 Promotes Tumor Metastasis by Targeting E-Cadherin in Nasopharyngeal Carcinoma

    doi: 10.1371/journal.ppat.1003974

    Figure Lengend Snippet: Depletion of endogenous miR-BART9 suppresses the migration and invasiveness of EBV-positive NPC cells. (A) LNA-modified anti-BART9 efficiently decreases the level of mature miR-BART9 in EBV-positive HK1-EBV and C666-1 cells. HK1-EBV and C666-1 cells were treated with a 12.5 nM concentration of an LNA-modified miR-BART9 antisense oligo (anti-BART9) or a scramble control (anti-Ctrl) for 48 hr. The expression level of miR-BART9 was determined via qPCR. (B) HK1-EBV cells were treated with anti-BART9 or anti-Ctrl for 24 hr and the plated for colony formation assays. Colony formation activity was determined via crystal violet staining after 11 days in culture. (C, D) Transwell migration assay (C) and Matrigel invasion assay (D) for HK1-EBV and C666-1 cells. Cells were treated with a 12.5 nM concentration of an LNA-modified miR-BART9 antisense oligo (anti-BART9) or a scramble control (anti-Ctrl) for 48 hr before the migration or invasion assay. Images of cells adhered to the lower surface of the filter insert from a representative experiment are shown. (Left panel). The numbers of migratory or invasive cells were quantified using image J and expressed as the fold change relative to the appropriate cell line (bar graphs). (E) Expression levels of LMP1, LMP2A and EBNA1 in HK1-EBV and C666-1 cells treated with a 12.5 nM concentration of an LNA-modified miR-BART9 antisense oligo (anti-BART9) or a scramble control (anti-Ctrl). Total RNA was collected 48 hr after transfection and mRNA levels were determined via qPCR. The data were normalized to cellular EEF1A1 levels and expressed as the fold change relative to the appropriate cell line. The bar graphs in (B), (C), (D), (E) show means ± SEM from three independent experiments and two-tailed Student's t-tests were performed (*, P

    Article Snippet: For mRNA quantification, 1 µg of total RNA was employed as a template for reverse transcription using an oligo dT primer and Superscript III (Invitrogen).

    Techniques: Migration, Modification, Concentration Assay, Expressing, Real-time Polymerase Chain Reaction, Activity Assay, Staining, Transwell Migration Assay, Invasion Assay, Transfection, Two Tailed Test

    miR-BART9 promotes the migration and invasion of EBV-negative NPC cells. Transwell migration assay (A) and Matrigel invasion assay (B) for miR-BART9- or LacZ-expressing EBV-negative NPC cells. BM1, TW04 and HK1 cells were infected with the miR-BART9 (BART9) or control (LacZ) vector. Cells expressing miR-BART9 or LacZ were directly compared in terms of migratory activity or invasion activity in the same assay (Left panel). In a separate assay, cells expressing miR-BART9 were treated with a 12.5 nM concentration of an LNA-modified miR-BART9 antisense oligo (anti-BART9) or a scramble control (anti-Ctrl) for 48 hr before the migration or invasion assay (middle panel). Images of cells adhered to the lower surface of the filter insert from a representative experiment are shown. The numbers of migratory or invasive cells were quantified using image J and expressed as the fold change relative to the appropriate cell line (bar graphs). The data are expressed as the means ± SEM from three independent experiments and two-tailed Student's t-tests were performed (*, P

    Journal: PLoS Pathogens

    Article Title: The Epstein-Barr Virus-Encoded MicroRNA MiR-BART9 Promotes Tumor Metastasis by Targeting E-Cadherin in Nasopharyngeal Carcinoma

    doi: 10.1371/journal.ppat.1003974

    Figure Lengend Snippet: miR-BART9 promotes the migration and invasion of EBV-negative NPC cells. Transwell migration assay (A) and Matrigel invasion assay (B) for miR-BART9- or LacZ-expressing EBV-negative NPC cells. BM1, TW04 and HK1 cells were infected with the miR-BART9 (BART9) or control (LacZ) vector. Cells expressing miR-BART9 or LacZ were directly compared in terms of migratory activity or invasion activity in the same assay (Left panel). In a separate assay, cells expressing miR-BART9 were treated with a 12.5 nM concentration of an LNA-modified miR-BART9 antisense oligo (anti-BART9) or a scramble control (anti-Ctrl) for 48 hr before the migration or invasion assay (middle panel). Images of cells adhered to the lower surface of the filter insert from a representative experiment are shown. The numbers of migratory or invasive cells were quantified using image J and expressed as the fold change relative to the appropriate cell line (bar graphs). The data are expressed as the means ± SEM from three independent experiments and two-tailed Student's t-tests were performed (*, P

    Article Snippet: For mRNA quantification, 1 µg of total RNA was employed as a template for reverse transcription using an oligo dT primer and Superscript III (Invitrogen).

    Techniques: Migration, Transwell Migration Assay, Invasion Assay, Expressing, Infection, Plasmid Preparation, Activity Assay, Concentration Assay, Modification, Two Tailed Test

    Matrix switching during migration affects motility disproportionally to the nature of the matrix (a) Using Star MAts HEp3 cell migration was analyzed on uniform substrates of collagen (CN), fibronectin (FN), and laminin (LN). The migration rates are displayed in (b) as from CN onto CN, from FN onto FN, and so on. (c) Using Block MAts, two adjacent matrixes were created as described in Methods. Cells were seeded onto one matrix, cultured to confluence, and subsequently allowed to migrate onto the adjacent matrix. Images taken at 0, 8, and 16 hours document migration and the morphological changes visible when HEp3 cells switch from CN onto adjacent poly-D-lysine (PDL), FN, and LN, or vice versa. (b) The quantitation of migration rates across these interfaces revealed significant matrix-specific effects and unexpected migration deficiencies that could only be contributed to matrix switching and not the matrix itself (CN vs. FN). Images were taken on the Axiovert 135 microscope. Microscope and boxplot details are described in Methods. Scale bars, 50 μm.

    Journal: Biomaterials

    Article Title: Magnetically attachable stencils and the non-destructive analysis of the contribution made by the underlying matrix to cell migration.

    doi: 10.1016/j.biomaterials.2012.07.018

    Figure Lengend Snippet: Matrix switching during migration affects motility disproportionally to the nature of the matrix (a) Using Star MAts HEp3 cell migration was analyzed on uniform substrates of collagen (CN), fibronectin (FN), and laminin (LN). The migration rates are displayed in (b) as from CN onto CN, from FN onto FN, and so on. (c) Using Block MAts, two adjacent matrixes were created as described in Methods. Cells were seeded onto one matrix, cultured to confluence, and subsequently allowed to migrate onto the adjacent matrix. Images taken at 0, 8, and 16 hours document migration and the morphological changes visible when HEp3 cells switch from CN onto adjacent poly-D-lysine (PDL), FN, and LN, or vice versa. (b) The quantitation of migration rates across these interfaces revealed significant matrix-specific effects and unexpected migration deficiencies that could only be contributed to matrix switching and not the matrix itself (CN vs. FN). Images were taken on the Axiovert 135 microscope. Microscope and boxplot details are described in Methods. Scale bars, 50 μm.

    Article Snippet: Glass and t.c. plastic substrates, specifically 6- and 12-well plates (Costar 3516 and 3513, Corning) were coated with collagen (rat tail type I, 354249, BD Biosciences), human plasma fibronectin, murine laminin (L2020, Sigma), poly-D-lysine (P7280, Sigma), and FITC gelatin ( , Fisher).

    Techniques: Migration, Blocking Assay, Cell Culture, Quantitation Assay, Microscopy

    Stabilization of LSAT mRNA by A23187 does not involve new transcription. HeLa cells were transfected with pLSGC or pLSAT plasmids, treated with 5 μM actinomycin D for 30 min to block new transcription, then treated with 10 μM A23187 for the times indicated (lanes 1–6). In lane 7, transcription was blocked for 1 h with actinomycin D without A23187 treatment. Total cytoplasmic RNA was purified, equal amounts resolved by Northern analysis and hybridized to probes for S-antigen or GAPDH.

    Journal: Gene Expression

    Article Title: Calcium-Induced Stabilization of AU-Rich Short-Lived mRNAs Is a Common Default Response

    doi:

    Figure Lengend Snippet: Stabilization of LSAT mRNA by A23187 does not involve new transcription. HeLa cells were transfected with pLSGC or pLSAT plasmids, treated with 5 μM actinomycin D for 30 min to block new transcription, then treated with 10 μM A23187 for the times indicated (lanes 1–6). In lane 7, transcription was blocked for 1 h with actinomycin D without A23187 treatment. Total cytoplasmic RNA was purified, equal amounts resolved by Northern analysis and hybridized to probes for S-antigen or GAPDH.

    Article Snippet: Calcium ionophore A23187 and actinomycin D were obtained from Sigma Chemical Company.

    Techniques: Transfection, Blocking Assay, Purification, Northern Blot

    Half-life of LSAT and LSGC mRNAs in HeLa cells treated with A23187. Cells were transfected with pLSGC or pLSAT plasmids, treated with 5 μM actinomycin D for the times indicated to block transcription. A23187 (10 μM) was added with actinomycin D to deplete plates of LSAT mRNA for 3 h. (A) Total cytoplasmic RNA was isolated, equal amounts resolved by Northern analysis and hybridized to 32 P-labeled probes specific to the S-antigen or GAPDH coding region. Data were derived by averaging the densitometric plots of two independent trials. (B) Data are plotted relative to the untreated control mRNA (time 0), which was set to 1.0 for normalization.

    Journal: Gene Expression

    Article Title: Calcium-Induced Stabilization of AU-Rich Short-Lived mRNAs Is a Common Default Response

    doi:

    Figure Lengend Snippet: Half-life of LSAT and LSGC mRNAs in HeLa cells treated with A23187. Cells were transfected with pLSGC or pLSAT plasmids, treated with 5 μM actinomycin D for the times indicated to block transcription. A23187 (10 μM) was added with actinomycin D to deplete plates of LSAT mRNA for 3 h. (A) Total cytoplasmic RNA was isolated, equal amounts resolved by Northern analysis and hybridized to 32 P-labeled probes specific to the S-antigen or GAPDH coding region. Data were derived by averaging the densitometric plots of two independent trials. (B) Data are plotted relative to the untreated control mRNA (time 0), which was set to 1.0 for normalization.

    Article Snippet: Calcium ionophore A23187 and actinomycin D were obtained from Sigma Chemical Company.

    Techniques: Transfection, Blocking Assay, Isolation, Northern Blot, Labeling, Derivative Assay

    CD80 upregulation by ACs under conditions of functional or blocked phagocytosis. ( a – e ) RAW264.7 murine macrophages were exposed to ACs (human Jurkat 77 cells) at a ratio of 10 ACs per macrophage, for 16 hours in presence of vehicle, “Veh” ( a , b ) or cytochalasin D, “CytoD” ( c , d ), and CD80 expression was analyzed using flow cytometry. The cells were treated with vehicle or 2 μM cytochalasin D starting at 1 hour before addition of the ACs and continuing throughout the incubation period of the macrophages with the ACs. ( e ) Quantification of CD80 relative levels (expressed as ΔMFI (change in mean fluorescence intensity) relative to the Unstimulated control “Unstim”) of the CD80 histogram) upon exposure to ACs in presence of vehicle or cytochalasin D. *p

    Journal: Scientific Reports

    Article Title: Autoantigen-Harboring Apoptotic Cells Hijack the Coinhibitory Pathway of T Cell Activation

    doi: 10.1038/s41598-018-28901-0

    Figure Lengend Snippet: CD80 upregulation by ACs under conditions of functional or blocked phagocytosis. ( a – e ) RAW264.7 murine macrophages were exposed to ACs (human Jurkat 77 cells) at a ratio of 10 ACs per macrophage, for 16 hours in presence of vehicle, “Veh” ( a , b ) or cytochalasin D, “CytoD” ( c , d ), and CD80 expression was analyzed using flow cytometry. The cells were treated with vehicle or 2 μM cytochalasin D starting at 1 hour before addition of the ACs and continuing throughout the incubation period of the macrophages with the ACs. ( e ) Quantification of CD80 relative levels (expressed as ΔMFI (change in mean fluorescence intensity) relative to the Unstimulated control “Unstim”) of the CD80 histogram) upon exposure to ACs in presence of vehicle or cytochalasin D. *p

    Article Snippet: Chemicals were purchased from Sigma Aldrich: Lipopolysaccharide (from E. coli O111:B4), Sigma cat # LPS25; Actinomycin D, Sigma cat # A9415; Cytochalasin D, Sigma cat # C8273.

    Techniques: Functional Assay, Expressing, Flow Cytometry, Cytometry, Incubation, Fluorescence

    The Entry/Gateway ® cloning procedures for generating epitope-tagged fusion proteins using the P-element destination vectors . The procedures of Entry/Gateway ® cloning are illustrated in the diagram as two major steps. (1) In the first step, a fragment encoding the open reading frame (ORF) is inserted into an entry vector to generate entry clones. Two entry vectors were described in this study: ( i ) the pGWS which uses a TA-based method; ( ii ) pENTR/D-TOPO (Invitrogen) which uses a TOPO-based method. (2) In the second step, the ORF in the entry clone is recombined into one of the P-DEST vectors.

    Journal: BMC Cell Biology

    Article Title: An Entry/Gateway® cloning system for general expression of genes with molecular tags in Drosophila melanogaster

    doi: 10.1186/1471-2121-10-8

    Figure Lengend Snippet: The Entry/Gateway ® cloning procedures for generating epitope-tagged fusion proteins using the P-element destination vectors . The procedures of Entry/Gateway ® cloning are illustrated in the diagram as two major steps. (1) In the first step, a fragment encoding the open reading frame (ORF) is inserted into an entry vector to generate entry clones. Two entry vectors were described in this study: ( i ) the pGWS which uses a TA-based method; ( ii ) pENTR/D-TOPO (Invitrogen) which uses a TOPO-based method. (2) In the second step, the ORF in the entry clone is recombined into one of the P-DEST vectors.

    Article Snippet: We compared the pGWS result with other LR reactions we performed with pENTR/D-TOPO (Invitrogen) entry clones, and the LR reaction efficiencies are very similar between pGWS-based and pENTR/D-TOPO-based entry clones (data not shown).

    Techniques: Clone Assay, Plasmid Preparation

    Increased levels of DNA damage in CSB mutant cells after a prolongued 5-azadC treatment (A–D) AA8 and UV61 cells were treated for 24 h with 5-azadC (2μM) washed and allowed to repair for 24h. Then were fixed and prepared for immunological staining of γ-H2AX (A-B), 53BP1 (C) and RAD51 (D). (E) In order to monitor residual DNA damage after a 24h treatment with 5-azadC, we performed micronucleus assay. Cells were treated with increasing doses of 5-azadC, washed and allowed to repair in cytochalasin B containing media for 18h. Cells were then fixed and scored for the number of micronuclei in binucleated cells. The means and SEM of three independent experiments are shown. Data were statistically analysed using Student's t -test. Data were considered statistically different when P

    Journal: Oncotarget

    Article Title: The Cockayne syndrome protein B is involved in the repair of 5-AZA-2′-deoxycytidine-induced DNA lesions

    doi: 10.18632/oncotarget.26189

    Figure Lengend Snippet: Increased levels of DNA damage in CSB mutant cells after a prolongued 5-azadC treatment (A–D) AA8 and UV61 cells were treated for 24 h with 5-azadC (2μM) washed and allowed to repair for 24h. Then were fixed and prepared for immunological staining of γ-H2AX (A-B), 53BP1 (C) and RAD51 (D). (E) In order to monitor residual DNA damage after a 24h treatment with 5-azadC, we performed micronucleus assay. Cells were treated with increasing doses of 5-azadC, washed and allowed to repair in cytochalasin B containing media for 18h. Cells were then fixed and scored for the number of micronuclei in binucleated cells. The means and SEM of three independent experiments are shown. Data were statistically analysed using Student's t -test. Data were considered statistically different when P

    Article Snippet: 5,6-Dichlorobenzimidazole 1-β-D-ribofuranoside (DRB), α-Amanitin and Cytochalasin B (Cyt B) were obtained from Sigma, dissolved in DMSO, aliquoted and stored at −80°C.

    Techniques: Mutagenesis, Staining

    Prolonged transcription inhibition potentiates 5-azadC-induced DNA damage in a CSB dependent manner (A–C) Cells were treated for 24h with 5-azadC (2μM), DRB (10μM) was added in the last 12h. Cells were then fixed and immunomarked for γ-H2AX (A), 53BP1 (B) and RAD51 (C). (D) DNA damage was also scored with the micronucleus test. Exponential cell cultures were treated 5-azadC ( 15μM) alone or in combination with DRB (20μM) or α-Amanitin (1μM) and allowed to recover in cytochalasin B containing media for 18h, fixed and the frequency of micronucleus was scored in binucleated cells. The means and SEM of three independent experiments are shown. Data were statistically analyzed using Student's t -test. Data were considered statistically different when P

    Journal: Oncotarget

    Article Title: The Cockayne syndrome protein B is involved in the repair of 5-AZA-2′-deoxycytidine-induced DNA lesions

    doi: 10.18632/oncotarget.26189

    Figure Lengend Snippet: Prolonged transcription inhibition potentiates 5-azadC-induced DNA damage in a CSB dependent manner (A–C) Cells were treated for 24h with 5-azadC (2μM), DRB (10μM) was added in the last 12h. Cells were then fixed and immunomarked for γ-H2AX (A), 53BP1 (B) and RAD51 (C). (D) DNA damage was also scored with the micronucleus test. Exponential cell cultures were treated 5-azadC ( 15μM) alone or in combination with DRB (20μM) or α-Amanitin (1μM) and allowed to recover in cytochalasin B containing media for 18h, fixed and the frequency of micronucleus was scored in binucleated cells. The means and SEM of three independent experiments are shown. Data were statistically analyzed using Student's t -test. Data were considered statistically different when P

    Article Snippet: 5,6-Dichlorobenzimidazole 1-β-D-ribofuranoside (DRB), α-Amanitin and Cytochalasin B (Cyt B) were obtained from Sigma, dissolved in DMSO, aliquoted and stored at −80°C.

    Techniques: Inhibition

    p53 promotes oxidative phosphorylation (A) The fluorescence lifetime histogram on the phasor plot shows the distribution of lifetimes of autofluorescent NADH. A color spectrum indicates the bound fraction of NADH. (B) Fluorescence intensity images of GFP of an EGFP cell and p53-GFP cell (left panels). Normalized fluorescence intensity images of NADH (middle panels) and pseudo-colored FLIM images (right panels) of H1299 lung cancer cells transfected with EGFP and p53-GFP. Pseudo-coloring corresponds to the phasor position according to the color spectrum in Figure 1A. Scale bar = 10μm. (C) Bar graphs quantifying the bound fraction of NADH in the nucleus (left) and cytoplasm (right). (D) Percent change in bound fraction of NADH in the nucleus (left) and cytoplasm (right) after treatment with DMSO, rotenone and antimycin A (R+AA), or 2-deoxy-D-glucose and dichloroacetate (DG+DCA). * p

    Journal: Biochemical and biophysical research communications

    Article Title: Absence of REV3L promotes p53-regulated cancer cell metabolism in cisplatin-treated lung carcinoma cells

    doi: 10.1016/j.bbrc.2018.01.026

    Figure Lengend Snippet: p53 promotes oxidative phosphorylation (A) The fluorescence lifetime histogram on the phasor plot shows the distribution of lifetimes of autofluorescent NADH. A color spectrum indicates the bound fraction of NADH. (B) Fluorescence intensity images of GFP of an EGFP cell and p53-GFP cell (left panels). Normalized fluorescence intensity images of NADH (middle panels) and pseudo-colored FLIM images (right panels) of H1299 lung cancer cells transfected with EGFP and p53-GFP. Pseudo-coloring corresponds to the phasor position according to the color spectrum in Figure 1A. Scale bar = 10μm. (C) Bar graphs quantifying the bound fraction of NADH in the nucleus (left) and cytoplasm (right). (D) Percent change in bound fraction of NADH in the nucleus (left) and cytoplasm (right) after treatment with DMSO, rotenone and antimycin A (R+AA), or 2-deoxy-D-glucose and dichloroacetate (DG+DCA). * p

    Article Snippet: For inhibition of glycolysis, cells were treated with 20 mM 2-deoxy-D-glucose and 5 mM dichloroacetate (Sigma-Aldrich, St. Louis, MO) for 6 hours prior to imaging.

    Techniques: Fluorescence, Transfection

    Dendritic release requires L-type Ca 2+ channels. A , Dendritic release responses to AMPA or NMDA in the presence and absence of IEM1460 (100 µM) or nimodipine (3 µM). n ≥ 5. ****p

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    Article Title: Action potential-independent and pharmacologically unique vesicular serotonin release from dendrites

    doi: 10.1523/JNEUROSCI.0020-12.2012

    Figure Lengend Snippet: Dendritic release requires L-type Ca 2+ channels. A , Dendritic release responses to AMPA or NMDA in the presence and absence of IEM1460 (100 µM) or nimodipine (3 µM). n ≥ 5. ****p

    Article Snippet: Parachloroamphetamine (Sigma) was also applied at 32° C. For stimulation with ionotropic glutamate receptor agonists, AMPA (Sigma, 10 µM) or NMDA (Sigma, 50 µM) were applied to slices in room temperature n-aCSF for 1 minute.

    Techniques:

    Comparative proteomic analysis of vehicle- and endorepellin-treated human endothelial cells . Representative 2-D protein profiles from vehicle- and endorepellin-treated endothelial cells are depicted with protein separation according to isoelectric point (pI, x-axis) and molecular weight (kDa, y-axis). Encircled protein spots correspond to the five proteins differentially expressed in response to endorepellin treatment. Compare calreticulin (1), β-actin (2), chaperonin/Hsp60 (3), vimentin (4) and prolyl 4-hydorxylase, β subunit (5), spot intensities in the two sets of samples. The spots under the bracket represent serum albumin by LC-MS/MS.

    Journal: Proteome Science

    Article Title: Proteomic profiling of endorepellin angiostatic activity on human endothelial cells

    doi: 10.1186/1477-5956-6-7

    Figure Lengend Snippet: Comparative proteomic analysis of vehicle- and endorepellin-treated human endothelial cells . Representative 2-D protein profiles from vehicle- and endorepellin-treated endothelial cells are depicted with protein separation according to isoelectric point (pI, x-axis) and molecular weight (kDa, y-axis). Encircled protein spots correspond to the five proteins differentially expressed in response to endorepellin treatment. Compare calreticulin (1), β-actin (2), chaperonin/Hsp60 (3), vimentin (4) and prolyl 4-hydorxylase, β subunit (5), spot intensities in the two sets of samples. The spots under the bracket represent serum albumin by LC-MS/MS.

    Article Snippet: Protein was quantified using the 2-D Quant Kit (GE Healthcare), collected and stored at -80°C.

    Techniques: Molecular Weight, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    Immunoblotting supports the proteomic analysis and indicates relative changes of various endothelial cell proteins evoked by endorepellin treatment . (A) Close up view of two representative 2-D gels, depicting the decline in calreticulin and β-actin levels. Notice that the major actin isoforms also show reduced staining intensity in response to endorepellin treatment. (B) Immunoblotting of control and endorepellin-treated endothelial cell lysates using an antibody against human calreticulin. Equal amounts of total proteins were loaded. (C) Immunoblot analysis of β-actin levels following treatment with ~200 nM endorepellin for the designated time intervals. (D) The kinetic data for β-actin expression levels were derived from immunoblotting data as presented in C. Essentially HUVEC β-actin expression levels were examined by Western blot following exposure to endorepellin for the indicated time points. β-actin levels were quantified over similar amounts of protein loading and graphed as percent β-actin levels (based upon time point 0) versus exposure to endorepellin treatment (hours). The data were graphed in SigmaPlot v9 and fit by non-linear regression analysis. Each data point represents the mean ± S.E.M. from three experiments. The T1/2 of ~45 min. represents the time to reduce β-actin levels by 50% as compared to the control. (E) Immunoblot analysis of vimentin and prolyl 4-hydroxylase, β subunit protein levels following endorepellin treatment for the indicated time points. GAPDH is shown as loading control. All verification experiments presented in panels C-E utilized new samples of endorepellin-treated HUVEC lysates, separate from those used in proteomic analysis.

    Journal: Proteome Science

    Article Title: Proteomic profiling of endorepellin angiostatic activity on human endothelial cells

    doi: 10.1186/1477-5956-6-7

    Figure Lengend Snippet: Immunoblotting supports the proteomic analysis and indicates relative changes of various endothelial cell proteins evoked by endorepellin treatment . (A) Close up view of two representative 2-D gels, depicting the decline in calreticulin and β-actin levels. Notice that the major actin isoforms also show reduced staining intensity in response to endorepellin treatment. (B) Immunoblotting of control and endorepellin-treated endothelial cell lysates using an antibody against human calreticulin. Equal amounts of total proteins were loaded. (C) Immunoblot analysis of β-actin levels following treatment with ~200 nM endorepellin for the designated time intervals. (D) The kinetic data for β-actin expression levels were derived from immunoblotting data as presented in C. Essentially HUVEC β-actin expression levels were examined by Western blot following exposure to endorepellin for the indicated time points. β-actin levels were quantified over similar amounts of protein loading and graphed as percent β-actin levels (based upon time point 0) versus exposure to endorepellin treatment (hours). The data were graphed in SigmaPlot v9 and fit by non-linear regression analysis. Each data point represents the mean ± S.E.M. from three experiments. The T1/2 of ~45 min. represents the time to reduce β-actin levels by 50% as compared to the control. (E) Immunoblot analysis of vimentin and prolyl 4-hydroxylase, β subunit protein levels following endorepellin treatment for the indicated time points. GAPDH is shown as loading control. All verification experiments presented in panels C-E utilized new samples of endorepellin-treated HUVEC lysates, separate from those used in proteomic analysis.

    Article Snippet: Protein was quantified using the 2-D Quant Kit (GE Healthcare), collected and stored at -80°C.

    Techniques: Staining, Expressing, Derivative Assay, Western Blot

    Nocodazole Does Not Inhibit rAAV2 Transduction ( a ) Microtubule and Golgi morphology. HeLa cells were preincubated in 1µM nocodazole for 4 hours and then fixed to observe the microtubule morphology (anti-beta-tubulin, Cy2, green) and Golgi localization (anti-p230, Cy3, red). Nuclei are in blue (DAPI). Scale bar = 25µm. ( b ) Percent GFP+ cells, normalized to no inhibitor control, as a function of nocodazole concentrations. After 4 hours preincubation with the nocodazole concentrations indicated, HeLa cells were transduced with rAAV2-GFP at 10 4 gcp/cell. Twenty-four hours after transduction, the cells were harvested and stained with 7-amino-actinomycin D (7AAD) prior to FACS analysis for GFP. The GFP+ percentages were determined on the 7AAD− subset of a coherent population first defined by FSC/SSC. The %GFP populations were then normalized to a no inhibitor control represented by mean ± SD. ( c ) Normalized viability as a function of nocodazole concentrations. Viability was defined as FSC/SSC coherent, 7AAD− population, which was then normalized to a no inhibitor control.

    Journal: Virology

    Article Title: Effect of Inhibition of Dynein Function and Microtubule-Altering Drugs on AAV2 Transduction

    doi: 10.1016/j.virol.2007.05.009

    Figure Lengend Snippet: Nocodazole Does Not Inhibit rAAV2 Transduction ( a ) Microtubule and Golgi morphology. HeLa cells were preincubated in 1µM nocodazole for 4 hours and then fixed to observe the microtubule morphology (anti-beta-tubulin, Cy2, green) and Golgi localization (anti-p230, Cy3, red). Nuclei are in blue (DAPI). Scale bar = 25µm. ( b ) Percent GFP+ cells, normalized to no inhibitor control, as a function of nocodazole concentrations. After 4 hours preincubation with the nocodazole concentrations indicated, HeLa cells were transduced with rAAV2-GFP at 10 4 gcp/cell. Twenty-four hours after transduction, the cells were harvested and stained with 7-amino-actinomycin D (7AAD) prior to FACS analysis for GFP. The GFP+ percentages were determined on the 7AAD− subset of a coherent population first defined by FSC/SSC. The %GFP populations were then normalized to a no inhibitor control represented by mean ± SD. ( c ) Normalized viability as a function of nocodazole concentrations. Viability was defined as FSC/SSC coherent, 7AAD− population, which was then normalized to a no inhibitor control.

    Article Snippet: GFP expression was analyzed by FACS as follows: after trypsinization, cells were resuspended in 100 µl lx binding buffer with 5 µl (0.25 µg) of 7-amino-actinomycin D (7AAD, Annexin V-PE Apoptosis Detection Kit, BD Pharmingen, San Diego, CA).

    Techniques: Transduction, Staining, FACS

    FCM analysis of PB-derived VSELs. Erythrocytes were removed by hypotonic lysis and PB NCs were stained with fluorescein isothiocyanate-lineage markers (Lin), phycoerythrin-CD45 and allophycocyanin-CD133, CD34, or CXCR4 mAbs. (a) Left dot plot: 10 6  events ranging from 2 to 10  µ m were inclu ded in the P2 gate. Right histogram: Lin −  cells were included in the P1 analysis gate. Middle histogram: P1 gated cells stained with 7-aminoactinomycin D (7-AAD) were included in the P2 gate. Right histogram: Lin −  cells were included in the P3 gate and were analyzed for CD45 coexpression with CD133, CD34, or CXCR4 antigens. (b) Dot plots showing the size of analyzed CD133 + , CD34 + , or CXCR4 + , Lin − , CD45 −  VSELs (upper row) and CD133 + , CD34 + , or CXCR4 + , Lin − , CD45 +  HSC (lower row).

    Journal: Stem Cells International

    Article Title: Human Very Small Embryonic-Like Stem Cells Are Present in Normal Peripheral Blood of Young, Middle-Aged, and Aged Subjects

    doi: 10.1155/2016/7651645

    Figure Lengend Snippet: FCM analysis of PB-derived VSELs. Erythrocytes were removed by hypotonic lysis and PB NCs were stained with fluorescein isothiocyanate-lineage markers (Lin), phycoerythrin-CD45 and allophycocyanin-CD133, CD34, or CXCR4 mAbs. (a) Left dot plot: 10 6 events ranging from 2 to 10  µ m were inclu ded in the P2 gate. Right histogram: Lin − cells were included in the P1 analysis gate. Middle histogram: P1 gated cells stained with 7-aminoactinomycin D (7-AAD) were included in the P2 gate. Right histogram: Lin − cells were included in the P3 gate and were analyzed for CD45 coexpression with CD133, CD34, or CXCR4 antigens. (b) Dot plots showing the size of analyzed CD133 + , CD34 + , or CXCR4 + , Lin − , CD45 − VSELs (upper row) and CD133 + , CD34 + , or CXCR4 + , Lin − , CD45 + HSC (lower row).

    Article Snippet: Finally, 7-aminoactinomycin D (7-AAD; BD, Le Pont de Claix, France) was added to stain nucleated cells.

    Techniques: Derivative Assay, Lysis, Staining

    Mechanisms for KLRG1 + CD8 T cells suppressing tumors. ( a ) KLRG1 + CD8 T cells or KLRG1 − CD8 T cells were co-cultured with B16-GFP cells (green) at the E:T ratio of 5:1, and the killing process was captured by PE spinning disk live cell confocal microscope with a 60 × oil immersion lens. ( b ) KLRG1 + CD8 T cells or KLRG1 − CD8 T cells were co-cultured with B16-GFP cells at the E:T ratio of 5:1 for 24 hours. Then target cells were collected and stained with 7-AAD. Percentages of 7-AAD positive populations indicated the killing rates. ( c ) KLRG1 + CD8 T cells or KLRG1 − CD8 T cells were co-cultured with EL4 cells at the E:T ratio of 20:1 for 12 hours. Then target cells were collected and stained with 7-AAD. Percentages of 7-AAD positive populations indicated the killing rates. ( d ) KLRG1 + CD8 T cells and KLRG1 − CD8 T cells were co-cultured with EL4 cells at the E:T ratio of 5:1 and 20:1 for 24 h with or without 50ug/mL anti-FasL, 50ug/mL anti-TRAIL, and 50 μM Granzyme B inhibitor Z-AAD-CMK. Cytotoxicity against target cells was evaluated and shown. ( e ) In an in vitro matrigel invasion experiment, KLRG1 + CD8 T cells or KLRG1 − CD8 T cells were sorted and inoculated on the upper layer. After 24 hours, penetrated cells on the lower layer were collected and calculated. ( f – h ) Real-time PCR ( f , g ) was carried out to examine the gene expression of heparanase and p53, which were also confirmed by RNA-seq analysis. ( h ) In vitro experiments were performed in triplicates for three times.

    Journal: Scientific Reports

    Article Title: Allogeneic dendritic cells induce potent antitumor immunity by activating KLRG1+CD8 T cells

    doi: 10.1038/s41598-019-52151-3

    Figure Lengend Snippet: Mechanisms for KLRG1 + CD8 T cells suppressing tumors. ( a ) KLRG1 + CD8 T cells or KLRG1 − CD8 T cells were co-cultured with B16-GFP cells (green) at the E:T ratio of 5:1, and the killing process was captured by PE spinning disk live cell confocal microscope with a 60 × oil immersion lens. ( b ) KLRG1 + CD8 T cells or KLRG1 − CD8 T cells were co-cultured with B16-GFP cells at the E:T ratio of 5:1 for 24 hours. Then target cells were collected and stained with 7-AAD. Percentages of 7-AAD positive populations indicated the killing rates. ( c ) KLRG1 + CD8 T cells or KLRG1 − CD8 T cells were co-cultured with EL4 cells at the E:T ratio of 20:1 for 12 hours. Then target cells were collected and stained with 7-AAD. Percentages of 7-AAD positive populations indicated the killing rates. ( d ) KLRG1 + CD8 T cells and KLRG1 − CD8 T cells were co-cultured with EL4 cells at the E:T ratio of 5:1 and 20:1 for 24 h with or without 50ug/mL anti-FasL, 50ug/mL anti-TRAIL, and 50 μM Granzyme B inhibitor Z-AAD-CMK. Cytotoxicity against target cells was evaluated and shown. ( e ) In an in vitro matrigel invasion experiment, KLRG1 + CD8 T cells or KLRG1 − CD8 T cells were sorted and inoculated on the upper layer. After 24 hours, penetrated cells on the lower layer were collected and calculated. ( f – h ) Real-time PCR ( f , g ) was carried out to examine the gene expression of heparanase and p53, which were also confirmed by RNA-seq analysis. ( h ) In vitro experiments were performed in triplicates for three times.

    Article Snippet: Cells were harvested every 12 h and were incubated with 7-AAD (Molecular Probes, Invitrogen) at room temperature for 10 min.

    Techniques: Cell Culture, Microscopy, Staining, In Vitro, Real-time Polymerase Chain Reaction, Expressing, RNA Sequencing Assay

    Expression of HC29 protein and Western blotting. Gels were stained with Coomassie blue, and 15 µL samples were loaded per lane. Lane PM: Standard protein molecular weight marker, Lane 1: Extracts of Escherichia ( E. ) coli (BL21 strain) transformed with pET-28a empty expression vector before induction with isopropyl-β-D-thiogalactopyranoside (IPTG, negative control), Lane 2: Extracts of E. coli (BL21 strain) transformed with pET-28a empty expression vector after 5 h of induction with IPTG (negative control), Lane 3: Extracts of E. coli (BL21 strain) transformed with pET-28a/HC29 before induction with IPTG, Lanes 4~8: Extracts of E. coli (BL21 strain) transformed with pET-28a/HC29 after 1~5 h of induction with IPTG, Lane 9: Purified recombinant HC29 protein supernatants, Lane 10: HC29 natural protein in soluble proteins of Haemonchus contortus was identified using rat sera as a primary antibody.

    Journal: Journal of Veterinary Science

    Article Title: Cloning and characterization of a selenium-independent glutathione peroxidase (HC29) from adult Haemonchus contortus

    doi: 10.4142/jvs.2012.13.1.49

    Figure Lengend Snippet: Expression of HC29 protein and Western blotting. Gels were stained with Coomassie blue, and 15 µL samples were loaded per lane. Lane PM: Standard protein molecular weight marker, Lane 1: Extracts of Escherichia ( E. ) coli (BL21 strain) transformed with pET-28a empty expression vector before induction with isopropyl-β-D-thiogalactopyranoside (IPTG, negative control), Lane 2: Extracts of E. coli (BL21 strain) transformed with pET-28a empty expression vector after 5 h of induction with IPTG (negative control), Lane 3: Extracts of E. coli (BL21 strain) transformed with pET-28a/HC29 before induction with IPTG, Lanes 4~8: Extracts of E. coli (BL21 strain) transformed with pET-28a/HC29 after 1~5 h of induction with IPTG, Lane 9: Purified recombinant HC29 protein supernatants, Lane 10: HC29 natural protein in soluble proteins of Haemonchus contortus was identified using rat sera as a primary antibody.

    Article Snippet: Expression was induced with isopropyl-β-D-thiogalactopyranoside (Sigma, USA) to a final concentration of 1 mM.

    Techniques: Expressing, Western Blot, Staining, Molecular Weight, Marker, Transformation Assay, Positron Emission Tomography, Plasmid Preparation, Negative Control, Purification, Recombinant

    Deoxycholic acid does not disrupt endothelial tight junctions via known signal transduction pathways. Confluent monolayers of brain microvessel endothelial cells were treated for 24 hours with vehicle or deoxycholic acid in either the presence or absence

    Journal: Digestive and liver disease : official journal of the Italian Society of Gastroenterology and the Italian Association for the Study of the Liver

    Article Title: Bile acids permeabilize the blood brain barrier after bile duct ligation

    doi: 10.1016/j.dld.2014.01.159

    Figure Lengend Snippet: Deoxycholic acid does not disrupt endothelial tight junctions via known signal transduction pathways. Confluent monolayers of brain microvessel endothelial cells were treated for 24 hours with vehicle or deoxycholic acid in either the presence or absence

    Article Snippet: To assess the effects of bile acid treatment on the phosphorylation of occludin, RBMEC’s were treated with chenodeoxycholic acid or deoxycholic acid (100 μM) for 4 hr in the presence or absence of the Rac1 inhibitor (NSC23766; 50 μM [ ], EMD Millipore, Billerica, MA).

    Techniques: Transduction

    Chenodeoxycholic acid and deoxycholic acid disrupts endothelial tight junctions. (A) Confluent monolayers of brain microvessel endothelial cells were treated with either vehicle, chenodeoxycholic acid or deoxycholic acid and the tight junction protein

    Journal: Digestive and liver disease : official journal of the Italian Society of Gastroenterology and the Italian Association for the Study of the Liver

    Article Title: Bile acids permeabilize the blood brain barrier after bile duct ligation

    doi: 10.1016/j.dld.2014.01.159

    Figure Lengend Snippet: Chenodeoxycholic acid and deoxycholic acid disrupts endothelial tight junctions. (A) Confluent monolayers of brain microvessel endothelial cells were treated with either vehicle, chenodeoxycholic acid or deoxycholic acid and the tight junction protein

    Article Snippet: To assess the effects of bile acid treatment on the phosphorylation of occludin, RBMEC’s were treated with chenodeoxycholic acid or deoxycholic acid (100 μM) for 4 hr in the presence or absence of the Rac1 inhibitor (NSC23766; 50 μM [ ], EMD Millipore, Billerica, MA).

    Techniques:

    Bile acids increase the phosphorylation of occludin via a Rac1-dependent mechanism (A) Brain microvessel endothelial cells were treated with vehicle, chenodeoxycholic acid or deoxycholic acid for 1 hour and phosphorylated occludin levels were assessed

    Journal: Digestive and liver disease : official journal of the Italian Society of Gastroenterology and the Italian Association for the Study of the Liver

    Article Title: Bile acids permeabilize the blood brain barrier after bile duct ligation

    doi: 10.1016/j.dld.2014.01.159

    Figure Lengend Snippet: Bile acids increase the phosphorylation of occludin via a Rac1-dependent mechanism (A) Brain microvessel endothelial cells were treated with vehicle, chenodeoxycholic acid or deoxycholic acid for 1 hour and phosphorylated occludin levels were assessed

    Article Snippet: To assess the effects of bile acid treatment on the phosphorylation of occludin, RBMEC’s were treated with chenodeoxycholic acid or deoxycholic acid (100 μM) for 4 hr in the presence or absence of the Rac1 inhibitor (NSC23766; 50 μM [ ], EMD Millipore, Billerica, MA).

    Techniques:

    CT-facilitated reversal of LTP is NMDA receptor-dependent. Stable LTP was induced with HFS ( white arrow ) in animals injected with the NMDA receptor antagonist D-AP5 (100 nmol; black arrowhead ) 5 min before the administration of CT 105 (1 pmol) and 5 min

    Journal: The Journal of Neuroscience

    Article Title: Use-Dependent Effects of Amyloidogenic Fragments of β-Amyloid Precursor Protein on Synaptic Plasticity in Rat Hippocampus In Vivo

    doi: 10.1523/JNEUROSCI.21-04-01327.2001

    Figure Lengend Snippet: CT-facilitated reversal of LTP is NMDA receptor-dependent. Stable LTP was induced with HFS ( white arrow ) in animals injected with the NMDA receptor antagonist D-AP5 (100 nmol; black arrowhead ) 5 min before the administration of CT 105 (1 pmol) and 5 min

    Article Snippet: D-AP5 (100 nmol) was injected intracerebroventricularly 5 min post-HFS, and CT (1 pmol) was administered 5 min later.

    Techniques: Injection

    Cetuximab modulates macrophage polarization in an AOM/DSS mouse model A. Establishment of the AOM/DSS mouse model. AOM was injected intraperitoneally at 12.5 mg/kg body weight. After one week, mice were given drinking water containing 2.5% DSS for 5 days followed by 16 days of regular drinking water. After two cycles of DSS treatment, cetuximab (1 mg/mouse, twice a week) was injected intraperitoneally for a month, and the mice were then sacrificed. B. Representative images of colon tumors in normal (right), AOM/DSS (2AD) (middle), and cetuximab-treated AOM/DSS mice (2AD+cetu) (left). C. Tumor quantification. Cetuximab treatment (2AD + cetu) reduced tumor numbers compared to 2AD mice. D. p-EGFR (Y1068), EGFR, Arg1, and iNOS protein levels in normal mice, 2AD and 2AD+cetu mouse tumors were detected by Western blot. All experiments were repeated three times. E. Representative photomicrographs of immunostaining for p-EGFR (Y1068), PCNA, F4/80, Arg1, and iNOS. Scale bars: 25 μm. F. M1 marker (IL-12, iNOS) and M2 marker (IL-4, IL-10, Arg1) mRNA levels in normal mouse colon tissues, 2AD and 2AD+cetu mouse tumor tissues were evaluated by q-PCR. G. Percentages of CD11b + /F4/80 + and F4/80 + /CD206 + cells in normal, 2AD, and 2AD+cetu mice colon tissues were detected by flow cytometry. Colon tissues were cut into small pieces (1-2 mm) and incubated with collagenase D (1- mg/mL), dispase II (1 mg/mL), and DNase I (100 μg/mL) for 30-45 min in a shaking incubator at 37°C, and single-cell suspensions were then incubated with antibodies. Bars represent means ± SD (n = 3) for each treatment. * p

    Journal: Oncotarget

    Article Title: Polarization of macrophages in the tumor microenvironment is influenced by EGFR signaling within colon cancer cells

    doi: 10.18632/oncotarget.12207

    Figure Lengend Snippet: Cetuximab modulates macrophage polarization in an AOM/DSS mouse model A. Establishment of the AOM/DSS mouse model. AOM was injected intraperitoneally at 12.5 mg/kg body weight. After one week, mice were given drinking water containing 2.5% DSS for 5 days followed by 16 days of regular drinking water. After two cycles of DSS treatment, cetuximab (1 mg/mouse, twice a week) was injected intraperitoneally for a month, and the mice were then sacrificed. B. Representative images of colon tumors in normal (right), AOM/DSS (2AD) (middle), and cetuximab-treated AOM/DSS mice (2AD+cetu) (left). C. Tumor quantification. Cetuximab treatment (2AD + cetu) reduced tumor numbers compared to 2AD mice. D. p-EGFR (Y1068), EGFR, Arg1, and iNOS protein levels in normal mice, 2AD and 2AD+cetu mouse tumors were detected by Western blot. All experiments were repeated three times. E. Representative photomicrographs of immunostaining for p-EGFR (Y1068), PCNA, F4/80, Arg1, and iNOS. Scale bars: 25 μm. F. M1 marker (IL-12, iNOS) and M2 marker (IL-4, IL-10, Arg1) mRNA levels in normal mouse colon tissues, 2AD and 2AD+cetu mouse tumor tissues were evaluated by q-PCR. G. Percentages of CD11b + /F4/80 + and F4/80 + /CD206 + cells in normal, 2AD, and 2AD+cetu mice colon tissues were detected by flow cytometry. Colon tissues were cut into small pieces (1-2 mm) and incubated with collagenase D (1- mg/mL), dispase II (1 mg/mL), and DNase I (100 μg/mL) for 30-45 min in a shaking incubator at 37°C, and single-cell suspensions were then incubated with antibodies. Bars represent means ± SD (n = 3) for each treatment. * p

    Article Snippet: Briefly, colon tissues were cut into small pieces (1-2 mm) and incubated in 10 mL PRMI 1640 medium with 10 mM HEPES and 5% fetal bovine serum containing 10 mg (1 mg/mL) collagenase D (Sigma-Aldrich), 10 mg (1 mg/mL) dispase II (Roche, Germany), and 100 μL 10 mg/ml DNase I (100 μg/mL) (Sigma-Aldrich) for 30-45 min in a shaking incubator at 37°C.

    Techniques: Injection, Mouse Assay, Western Blot, Immunostaining, Marker, Polymerase Chain Reaction, Flow Cytometry, Cytometry, Incubation