Journal: BMC Neuroscience

Article Title: Functional crosstalk in culture between macrophages and trigeminal sensory neurons of a mouse genetic model of migraine

doi: 10.1186/1471-2202-13-143

Figure Lengend Snippet: Activated phenotype of trigeminal ganglion macrophages in vitro. A , Examples of fluorescence microscopy images of cultures from WT or KI trigeminal ganglia immunostained with antibodies against Iba1 (red), β˜-tubulin III (green) and counterstained with DAPI (blue). MФ. indicates co-culturing with known number of host macrophages, while only MФ .indicates pure peritoneal macrophage cultures. Scale bar: 50 μm. B , Histograms quantify the number of Iba1-positive cells in the ROI (640 × 480 μm) from WT or R129Q KI ganglion cultures in control condition and after MФ. addition; n = 3 petri dishes for each group run in parallel, * p < 0.05, ** p < 0.01. MФ .# p < 0.01 vs all conditions. C , Histograms quantify the average cell area (expressed in μm 2 ) for data shown in B , * p < 0.05, ** p < 0.01. D , P2X3 mRNA levels in co-cultures (WT+MФ, KI+MФ) expressed as fold increase in comparison to their controls (WT or R192Q KI cultures) and normalized versus β-tubulin mRNA levels (n = 3 petri dishes for each group). E , Example of western blots shows similar levels of P2X3 receptor expression in lysates from WT or R192Q KI trigeminal cultures (lanes 1 and 3), and in co-cultures (WT+MФ and KI+MФ, lanes 2 and 4). Lysates from MФ only (lane 5) show no signal. β-Tubulin III and actin are shown as loading control (bottom row). F , Histograms show mean values (optical density, AUs) of P2X3 subunits obtained in western blot experiments normalized over β-tubulin III signals; n = 4 petri dishes.

Article Snippet: Western blotting was performed as described earlier [ , ], using antibodies against anti-P2X3 (1:300; Alomone, Jerusalem, Israel), anti-β-tubulin III (1:2.000; Sigma) or anti-actin (1:3.000; Sigma).

Techniques: In Vitro, Fluorescence, Microscopy, Control, Comparison, Western Blot, Expressing

Journal: BMC Neuroscience

Article Title: Functional crosstalk in culture between macrophages and trigeminal sensory neurons of a mouse genetic model of migraine

doi: 10.1186/1471-2202-13-143

Figure Lengend Snippet: Neuronal P2X3 receptor-mediated responses in basal conditions or in the presence of host macrophages. A , Representative examples of currents induced by application of α,β-meATP (10 μM, 2 s; horizontal bar) to trigeminal neurons from WT ( n = 15 neurons) or R192Q KI ( n = 17) cultures in standard conditions (left traces) or when co-cultured with macrophages (WT+MФ, . n = 24; KI+MФ, . n = 20. Note that macrophage co-culturing increases P2X3-mediated responses from WT neurons. Average data are plotted in B . * p < 0.05; ** p < 0.01. C, Rise time (left; expressed as time from 10 to 90% of peak amplitude), desensitization onset (middle; expressed as the first time constant, τ fast , of current decay). Values are from 13-23 neurons. Recovery from desensitization (right; expressed as % of control amplitude in a paired pulse agonist application) was faster for WT+MФ vs WT; * p = 0.007. All responses were evoked by α,β-meATP (10 μM, 2 s). D , ATP medium content measured 5 h in WT or KI trigeminal neuron-macrophage co-cultures. Basal ATP levels present in culture macrophages are also shown. Data are expressed as fractional increase with respect to neuronal WT or KI cultures. n = 2, * p < 0.05.

Article Snippet: Western blotting was performed as described earlier [ , ], using antibodies against anti-P2X3 (1:300; Alomone, Jerusalem, Israel), anti-β-tubulin III (1:2.000; Sigma) or anti-actin (1:3.000; Sigma).

Techniques: Cell Culture, Control

Journal: Nature Communications

Article Title: RNF168 and USP10 regulate topoisomerase IIα function via opposing effects on its ubiquitylation

doi: 10.1038/ncomms12638

Figure Lengend Snippet: ( a ) Identification of RNF168-associated proteins. A representative SDS–polyacrylamide gel electrophoresis of Flag-RNF168-associated proteins. Flag-tagged RNF168 was transfected in HEK293T cells and pull-down analysis was performed 48 h later. Protein bands were detected by silver staining. Protein bands were identified by mass spectrometry analysis following in-gel protease digestion. ( b ) HEK293T cells were transfected as indicated with HA-tagged RNF168 and Flag-TOP2α expression vectors. Cells were lysed and IP was performed using anti-Flag antibody. The resulting precipitates were subjected to IB analysis with the indicated antibodies. WCL, whole-cell lysate. ( c ) TOP2α, RNF168 and IgG (control) immunoprecipitates from HEK293T cells were examined by IB as indicated. ( b , c ) Data are representative of three independent experiments. ( d ) Cells treated with EdU were used for detection of localization patterns of TOP2α (Alexa Fluor 488) and RNF168 (Alexa Fluor 594) using confocal microscopy. Scale bar, 20 μm.

Article Snippet: After washing with 3% BSA in PBS, the sheep polyclonal against RNF168 (1:2,000; R&D systems cat# AF7217) and the rabbit polyclonal against TOP2α (Abcam) were detected using their respective secondary antibodies.

Techniques: Polyacrylamide Gel Electrophoresis, Transfection, Silver Staining, Mass Spectrometry, Expressing, Control, Confocal Microscopy

Journal: Nature Communications

Article Title: RNF168 and USP10 regulate topoisomerase IIα function via opposing effects on its ubiquitylation

doi: 10.1038/ncomms12638

Figure Lengend Snippet: ( a ) Representative agarose gel of in vitro kinetoplast DNA-based decatenation assays performed for 10 min with different amount of nuclear extracts from WT and Rnf168 −/− MEFs. Catenated and decatenated kDNA were separated by electrophoresis using 1% agarose gel. IB show Top2α's level in the total nuclear extracts used for this assay. ( b ) Representative data of the mitotic inhibition assay of decatenation G2 checkpoint in WT and Rnf168 −/− primary MEFs. Cells were treated with DMSO or ICRF-193 for 15 min and then incubated in culture media for an additional 2 h. The fraction of mitotic cells (pHH3 + ) was determined by flow cytometry. ( c ) Bar graphs represent the mean inhibition of mitotic index 2 h post ICRF-193 treatment of passage 1 primary MEFs (% pHH3 + cells post ICRF-193 treatment compared with DMSO-treated controls). * P <0.05. ( d ) Bar graphs represent the mean inhibition of mitotic index 2 h post ICRF-193 treatment of RIDDLE cells reconstituted with either HA-RNF168 or HA-empty vector as in c . * P <0.05. ( e ) Analysis of decatenation G2 checkpoint of Rnf168 −/− and WT 3T3 MEFs using the mitotic entry assay. Percentage of pHH3 + cells is shown at the indicated time post-treatment with colcemid in the presence of DMSO or ICRF-193. * P <0.05; Rnf168 −/− MEFs compared with WT MEFs 6 h post-ICRF-193 treatment. ( f ) Bar graphs represent the mean fraction of pHH3 + WT and Rnf168 −/− MEFs evading G2 arrest 6 h post-treatment with ICRF-193 compared with DMSO-treated cells as in e . * P <0.05. ( g ) Mitotic entry assay of decatenation G2 checkpoint in human RIDDLE cells reconstituted with HA-empty vector or HA-RNF168. Percentage of pHH3 + cells is shown at the indicated times post-treatment with colcemid with or without ICRF-193. * P <0.05, RIDDLE cells reconstituted with HA-empty vector compared with RIDDLE cells reconstituted with HA-RNF168 at 6 h post-ICRF-193 treatment. ( h ) Bar graphs represent the mean fraction of RIDDLE cells (reconstituted with HA-empty vector compared with those reconstituted with HA-RNF168) evading G2 arrest 6 h post ICRF-193 treatment compared with DMSO-treated controls as in f . * P <0.05. Three independent experiments in triplicates unless specified. Error bars in c , d , f and h represent mean±s.e.m.

Article Snippet: After washing with 3% BSA in PBS, the sheep polyclonal against RNF168 (1:2,000; R&D systems cat# AF7217) and the rabbit polyclonal against TOP2α (Abcam) were detected using their respective secondary antibodies.

Techniques: Agarose Gel Electrophoresis, In Vitro, Electrophoresis, Inhibition, Incubation, Flow Cytometry, Plasmid Preparation

Journal: Nature Communications

Article Title: RNF168 and USP10 regulate topoisomerase IIα function via opposing effects on its ubiquitylation

doi: 10.1038/ncomms12638

Figure Lengend Snippet: ( a – d ) Sensitivity of WT and Rnf168 −/− MEFs to ICRF-193 ( a , b ) or etoposide ( c , d ) was determined using clonogenic assays. ( e , f ) Sensitivity of the human breast cancer cell lines T47D and MDA-MB-231 to etoposide was determined using clonogenic assays. ( a , c , e ) Data are presented as the mean±s.e.m. ( a , c , n >4; e , n =4). * P <0.05 for Rnf168 −/− MEFs compared with WT MEFs and T47D and MDA-MB-231 cells knocked down for RNF168 (sh.RNF168) compared with their respective controls (Sh.Ctr: ShRNA control). ( b , d ) Representative pictures of dishes showing surviving WT and Rnf168 −/− colonies post ICRF-193 ( b ) or etoposide ( d ) treatment. ( f ) Representative pictures of dishes showing surviving colonies of etoposide treated T47D and MDA-MB-231 cells.

Article Snippet: After washing with 3% BSA in PBS, the sheep polyclonal against RNF168 (1:2,000; R&D systems cat# AF7217) and the rabbit polyclonal against TOP2α (Abcam) were detected using their respective secondary antibodies.

Techniques: shRNA, Control

Journal: Nature Communications

Article Title: RNF168 and USP10 regulate topoisomerase IIα function via opposing effects on its ubiquitylation

doi: 10.1038/ncomms12638

Figure Lengend Snippet: ( a ) RIDDLE cells reconstituted with HA-RNF168 or HA-empty vector, and control HA-RNF168-reconstituted RIDDLE cells with TOP2α knock down were lysed and WCL subjected to IP with anti-TOP2α or IgG (control) antibodies. Immunoprecipitates were blotted with the indicated antibodies to detect ubiquitylated TOP2α. ( b ) Human breast cancer cell lines T47D and MDA-MB-231 knocked down for RNF168 (Sh.RNF168) and their control expressing ShRNA control (sh.Ctr) were examined for their level of ubiquitylated TOP2α as in a . ( c ) Rnf168 −/− , Brca1 −/− and WT MEFs were lysed and subjected to IP with anti-Top2α or IgG (control) antibodies. IPs from WCL were blotted with the indicated antibodies. ( d ) HEK293T cells were transfected with RNF168 (WT or mutant Rnf168-C21S), Flag-TOP2α and HA-Ub vectors as indicated. WCL were subjected to IP with anti-Flag, and IB analysis was performed using anti-HA antibody to detect ubiquitylated Flag-TOP2α. ( e ) Nuclear extracts from RIDDLE cells reconstituted with HA-RNF168 or HA-empty vector were subjected to IP with anti-TOP2α or IgG (control) antibodies. Immunoprecipitates were blotted with the indicated antibodies against K63- and K48-Ub linkages. ( f ) In vitro ubiquitylation of recombinant TOP2α in the presence of recombinant RNF168 (500 ng for lane 5, 1 μg for lanes 2, 3 and 6 and 2 μg for lane7), UBE1 (E1), UBE2E2 (E2) and Ub. Nuc, nuclear extract; WCL, whole-cell lysate.

Article Snippet: After washing with 3% BSA in PBS, the sheep polyclonal against RNF168 (1:2,000; R&D systems cat# AF7217) and the rabbit polyclonal against TOP2α (Abcam) were detected using their respective secondary antibodies.

Techniques: Plasmid Preparation, Control, Knockdown, Expressing, shRNA, Transfection, Mutagenesis, In Vitro, Recombinant

Journal: Nature Communications

Article Title: RNF168 and USP10 regulate topoisomerase IIα function via opposing effects on its ubiquitylation

doi: 10.1038/ncomms12638

Figure Lengend Snippet: ( a ) WT and Rnf168 −/− MEFs, and Top2α knockdown control MEFs were fixed and stained with DAPI. Representative cells with defective chromosome segregation, as indicated by chromosome bridges (arrow head) and micronuclei (arrow) are shown. ( b ) Histograms show quantification of cells with chromosome bridges or micronuclei (mean±s.e.m., n =3). * P <0.05. ( c ) Histograms show the fraction of RIDDLE cells reconstituted with HA-RNF168 or HA-empty vector that display micronuclei (mean±s.e.m., n =3). * P <0.05. ( d ; upper panels) Representative metaphase spreads showing undercondensed chromosomes in WT MEFs 24 h post ICRF-193 treatment (positive control), and in Rnf168 −/− DMSO-treated MEFs. ( d , lower panels) Representative metaphase spreads of DMSO-treated Rnf168 −/− MEFs complemented with RNF168-WT (WT) or the E3 ligase deficient RNF168-C21S (C21S). ( e ) Histograms show quantification of abnormal metaphase spreads with entangled or undercondensed chromosomes from the indicated cells (mean±s.e.m., n =3). * P <0.05 compared with WT MEFs. ( f ) Histograms present the mean fraction of MEFs evading ICRF-193-induced G2 arrest as compared with DMSO-treated controls (mean±s.e.m., n =3). Data are shown for WT MEFs, mock infected Rnf168 −/− MEFs and Rnf168 −/− MEFs complemented with RNF168-WT or RNF168-C21S. 3T3 MEFs were used for these experiments. Data shown are for 6 h post-treatment with colcemid±4 μM ICRF-193. * P <0.05 compared with WT MEFs. ( g ) Clonogenic assay was used to determine sensitivity to ICRF-193 of mock infected WT and Rnf168 −/− MEFs, as well as Rnf168 −/− MEFs complemented with RNF168 (WT or C21S mutant). Data are presented as the mean±s.e.m. ( n =4). * P <0.05 compared with WT MEFs. Scale bar, 20 μm.

Article Snippet: After washing with 3% BSA in PBS, the sheep polyclonal against RNF168 (1:2,000; R&D systems cat# AF7217) and the rabbit polyclonal against TOP2α (Abcam) were detected using their respective secondary antibodies.

Techniques: Knockdown, Control, Staining, Plasmid Preparation, Positive Control, Infection, Clonogenic Assay, Mutagenesis

Journal: Nature Communications

Article Title: RNF168 and USP10 regulate topoisomerase IIα function via opposing effects on its ubiquitylation

doi: 10.1038/ncomms12638

Figure Lengend Snippet: ( a ) Nuclear (Nuc) and chromatin (Chr) fractions prepared from WT, Rnf168 −/− and Brca1 −/− MEFs were analysed by IB for chromatin occupancy of Top2α. Additional IBs were performed with the indicated antibodies as controls. H4, histone H4. ( b ) Nuclear and chromatin fractions were prepared from the human breast cancer cell lines T47D and MDA-MB-231 knocked down for RNF168 (Sh.RNF168) and their controls (Sh.Ctr) and analysed by IB for the chromatin occupancy of TOP2α as in a . ( c ) HEK293T were transfected with Flag-TOP2α along with RNF168 (+) or empty vector (−) and their nuclear and chromatin fractions were prepared and examined by IB using the indicated antibodies. ( d ) A representative agarose gel showing decatenation activity of soluble nuclear and chromatin extracts from 2 WT and 2 Rnf168 −/− MEFs. In vitro kinetoplast DNA-based decatenation assay was performed for 20 min with different amounts of nuclear and chromatin extracts, and catenated and decatenated kDNA were separated by electrophoresis. IB using anti-Top2α was performed to show the level of Top2α present in each sample.

Article Snippet: After washing with 3% BSA in PBS, the sheep polyclonal against RNF168 (1:2,000; R&D systems cat# AF7217) and the rabbit polyclonal against TOP2α (Abcam) were detected using their respective secondary antibodies.

Techniques: Transfection, Plasmid Preparation, Agarose Gel Electrophoresis, Activity Assay, In Vitro, Electrophoresis

Journal: Nature Communications

Article Title: RNF168 and USP10 regulate topoisomerase IIα function via opposing effects on its ubiquitylation

doi: 10.1038/ncomms12638

Figure Lengend Snippet: ( a , b ) HEK293T cells were transfected with Flag-RNF168 and HA-USP10 vectors ( a ) or Flag-TOP2α and HA-USP10 vectors ( b ) as indicated. Cells were lysed and IP was performed using anti-Flag and anti-HA antibodies. The resulting precipitates were subjected to IB analysis with the indicated antibodies. WCL, whole-cell lysate. ( c ) HEK293T cells were transfected with Flag-TOP2α, RNF168, HA-USP10 and Myc-Ub vectors, as indicated. IP using anti-Flag and WCL was subjected to anti-Ub IB analysis to detect TOP2α ubiquitylation. ( d ) WT MEFs with knockdown of Usp10 (Sh1 and Sh2) and WT controls were examined for the level of Top2α ubiquitylation. Top2α was immunoprecipitated from whole-cell extracts and examined by IB for its ubiquitylation level using anti-Ub. IP using IgG was used as a control. The indicated antibodies were used for IB. ( e ) HEK293T cells were transfected with Flag-TOP2α with or without RNF168, HA-Ub, USP10 and USP10-C424A as indicated. TOP2α chromatin occupancy in these cells was examined by IB using anti-Flag antibodies and chromatin fractions (Chr). IB analysis of the chromatin fractions is also shown for the indicated antibodies. ( f ) A simplified model of RNF168-mediated regulation of TOP2α ubiquitylation and decatenation function.

Article Snippet: After washing with 3% BSA in PBS, the sheep polyclonal against RNF168 (1:2,000; R&D systems cat# AF7217) and the rabbit polyclonal against TOP2α (Abcam) were detected using their respective secondary antibodies.

Techniques: Transfection, Knockdown, Immunoprecipitation, Control

Journal: Journal of Translational Medicine

Article Title: Development of patient-derived xenograft models from a spontaneously immortal low-grade meningioma cell line, KCI-MENG1

doi: 10.1186/s12967-015-0596-8

Figure Lengend Snippet: Immunostaining of original tumor, low passage, and high passage KCI-MENG1 cells. The original patient-derived tumor ( top row ) showed moderate and patchy immunoreactivity for epithelial membrane antigen (EMA); strong and diffuse immunostaining for progesterone receptor (PR); and a Ki-67 proliferative index of 2–3%. There was also strong immunostaining for N-cadherin and vimentin. KCI-MENG1-LP cells ( middle row ) and KCI-MENG1-HP cells ( bottom row ) maintained expression of EMA, N-cadherin, and vimentin but had significantly reduced PR expression compared to the original tumor. Whereas Ki-67 labeling was found in only a small number of cells in the original tumor and low passage cells, it was positive in virtually all P84 cells. Scale bar 50 µm.

Article Snippet: Primary antibodies used targeted the following proteins: EMA (cat.#247M-94), PR (cat.#323R-14), Ki-67 (cat.#275R-14), vimentin (cat.#347R-14; all from CellMarque, Rocklin, CA, USA), and N-cadherin (cat.#NBP1-48309, Novus Biologicals, Littleton, CO, USA).

Techniques: Immunostaining, Derivative Assay, Membrane, Expressing, Labeling

Journal: Journal of Translational Medicine

Article Title: Development of patient-derived xenograft models from a spontaneously immortal low-grade meningioma cell line, KCI-MENG1

doi: 10.1186/s12967-015-0596-8

Figure Lengend Snippet: Immunostaining of original patient tumor, low and high passage KCI-MENG1 cells, and subcutaneous xenograft tumor. The original patient-derived tumor showed moderate immunoreactivity for E-cadherin which was maintained in all in vitro and in vivo models. Scale bar 50 µm.

Article Snippet: Primary antibodies used targeted the following proteins: EMA (cat.#247M-94), PR (cat.#323R-14), Ki-67 (cat.#275R-14), vimentin (cat.#347R-14; all from CellMarque, Rocklin, CA, USA), and N-cadherin (cat.#NBP1-48309, Novus Biologicals, Littleton, CO, USA).

Techniques: Immunostaining, Derivative Assay, In Vitro, In Vivo

Journal: Journal of Translational Medicine

Article Title: Development of patient-derived xenograft models from a spontaneously immortal low-grade meningioma cell line, KCI-MENG1

doi: 10.1186/s12967-015-0596-8

Figure Lengend Snippet: Meningioma cell lines reported in the literature

Article Snippet: Primary antibodies used targeted the following proteins: EMA (cat.#247M-94), PR (cat.#323R-14), Ki-67 (cat.#275R-14), vimentin (cat.#347R-14; all from CellMarque, Rocklin, CA, USA), and N-cadherin (cat.#NBP1-48309, Novus Biologicals, Littleton, CO, USA).

Techniques: Southern Blot, Injection, Expressing, TRAP Assay, Activity Assay, Real-time Polymerase Chain Reaction

Journal: Journal of Translational Medicine

Article Title: Development of patient-derived xenograft models from a spontaneously immortal low-grade meningioma cell line, KCI-MENG1

doi: 10.1186/s12967-015-0596-8

Figure Lengend Snippet: Human meningioma mouse xenograft model KCI-MENG1-LPSX generated with the spontaneously immortal cell line KCI-MENG1-LP. Tumors from immunocompromised SCID mice were dissected ( a ) and the derivative cell line KCI-MENG1-LPSX CL was generated. The H&E staining of the mouse tumor revealed a pattern of moderately cellular meningothelial cells similar to the original patient tumor ( b ). The KCI-MENG1-LPSX CL cells were composed of the round-shaped cells similar to the high passage parent cell line KCI-MENG1-HP ( c ). The EMA, PR, and N-cadherin IHC of the mouse tumor highly resembled the original patient-derived tumor ( d top row ). The vimentin- and Ki-67-stained cells in the mouse tumor tissue were markedly more abundant and more intensely stained than in the original tumor ( d top row ). KCI-MENG1-LPSX CL cells displayed the same patterns of immunostaining as the high passage parent cell line KCI-MENG1-HP, including the loss of PR staining ( d bottom row ). Scale bar 50 µm.

Article Snippet: Primary antibodies used targeted the following proteins: EMA (cat.#247M-94), PR (cat.#323R-14), Ki-67 (cat.#275R-14), vimentin (cat.#347R-14; all from CellMarque, Rocklin, CA, USA), and N-cadherin (cat.#NBP1-48309, Novus Biologicals, Littleton, CO, USA).

Techniques: Generated, Staining, Derivative Assay, Immunostaining

Journal: PloS one

Article Title: A novel highly potent therapeutic antibody neutralizes multiple human chemokines and mimics viral immune modulation.

doi: 10.1371/journal.pone.0043332

Figure Lengend Snippet: Figure 1. Binding and in vitro activity of murine 18V4F hybridoma antibody. (A) ELISA binding of original 18V4F hybridoma antibody to a panel of chemokines (determined in triplicate, shown as mean +/2 standard deviation). Directly coated chemokines were used here for direct comparisons, however in other experiments CCL3 showed a significantly enhanced signal when biotinylated and coated on streptavidin plates. (B) Chemotaxis inhibition by 18V4F hybridoma antibody of CCR5-transfected Ba/F3 cells to 5 ng/mL of CCL3, CCL4, and CCL5. Data are representative of at least three similar experiments. All chemotaxis data are represented as a percent of maximum migration in the absence of inhibitors and is fit using a standard four parameter dose-response model (GraphPad). doi:10.1371/journal.pone.0043332.g001

Article Snippet: Control inhibitors included vCCI-Fc (produced at VLST – the Fc is from human IgG1 with mutations to prevent interactions with Fc receptors [38]) and commercial antibodies against CCL3 (R&D Systems #MAB270), CCL4 (R&D Systems #MAB271), and CCL5 (R&D Systems #MAB278) as well as IgG controls.

Techniques: Binding Assay, In Vitro, Activity Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation, Chemotaxis Assay, Inhibition, Transfection, Migration

Journal: PloS one

Article Title: A novel highly potent therapeutic antibody neutralizes multiple human chemokines and mimics viral immune modulation.

doi: 10.1371/journal.pone.0043332

Figure Lengend Snippet: Figure 2. Diagram of phage display selection strategy. Individual CDR libraries were sequentially panned against CCL3, CCL4 and CCL5. In step 1, phage libraries were combined with biotinylated CCL3 and bound to streptavidin beads. Bound phage were eluted, amplified, and subjected to panning against biotinylated CCL4 and CCL5 in steps 2 and 3, respectively. This process was repeated 4–5 times with increasing stringency to yield sequences with improved affinities. doi:10.1371/journal.pone.0043332.g002

Article Snippet: Control inhibitors included vCCI-Fc (produced at VLST – the Fc is from human IgG1 with mutations to prevent interactions with Fc receptors [38]) and commercial antibodies against CCL3 (R&D Systems #MAB270), CCL4 (R&D Systems #MAB271), and CCL5 (R&D Systems #MAB278) as well as IgG controls.

Techniques: Selection, Amplification

Journal: PloS one

Article Title: A novel highly potent therapeutic antibody neutralizes multiple human chemokines and mimics viral immune modulation.

doi: 10.1371/journal.pone.0043332

Figure Lengend Snippet: Figure 3. Chemotaxis inhibition by affinity matured 18V4F variants. Chemotaxis inhibition by humanized 18V4F Fab, d5 variant, d7 variant, d5d7, and a negative control Fab of CCR5-transfected Ba/F3 cells to 5 ng/mL of (A) CCL3, (B) CCL4, and (C) CCL5. Data are representative of at least two similar experiments. A loss in potency of humanized 18V4F Fab was observed compared with the 18V4F hybridoma shown in Figure 1b and is likely a result of both the humanization process and loss in avidity caused by switching from full IgG to Fab fragment. doi:10.1371/journal.pone.0043332.g003

Article Snippet: Control inhibitors included vCCI-Fc (produced at VLST – the Fc is from human IgG1 with mutations to prevent interactions with Fc receptors [38]) and commercial antibodies against CCL3 (R&D Systems #MAB270), CCL4 (R&D Systems #MAB271), and CCL5 (R&D Systems #MAB278) as well as IgG controls.

Techniques: Chemotaxis Assay, Inhibition, Variant Assay, Negative Control, Transfection

Journal: PloS one

Article Title: A novel highly potent therapeutic antibody neutralizes multiple human chemokines and mimics viral immune modulation.

doi: 10.1371/journal.pone.0043332

Figure Lengend Snippet: Figure 4. Comparison of vCCI and d5d7 binding epitopes. Competitive binding ELISA examining molecules that can disrupt the d5d7-CCL3 binding interaction using d5d7 as a homologous competitor and vCCI-Fc, commercial anti-CCL3 antibody, and control IgG as heterologous competitors. Data are representative of at least two similar experiments. Competition experiments were also completed to analyze the d5d7-CCL4 and d5d7-CCL5 binding interactions and similar binding competition was observed between d5d7 and vCCI-Fc (data not shown). doi:10.1371/journal.pone.0043332.g004

Article Snippet: Control inhibitors included vCCI-Fc (produced at VLST – the Fc is from human IgG1 with mutations to prevent interactions with Fc receptors [38]) and commercial antibodies against CCL3 (R&D Systems #MAB270), CCL4 (R&D Systems #MAB271), and CCL5 (R&D Systems #MAB278) as well as IgG controls.

Techniques: Comparison, Binding Assay, Enzyme-linked Immunosorbent Assay, Control

Journal: PloS one

Article Title: A novel highly potent therapeutic antibody neutralizes multiple human chemokines and mimics viral immune modulation.

doi: 10.1371/journal.pone.0043332

Figure Lengend Snippet: Figure 5. Inhibition of chemotaxis induced with mixtures of chemokines by MAb d5d7. Inhibition of chemotaxis of (A) CCR5 transfectants to a pool of recombinant CCL3, CCL4, and CCL5 and (B) CCR1 transfectants to a pool of CCL3 and CCL5, by MAb d5d7 antibody, vCCI-Fc, individual commercial anti-chemokine antibodies (anti-CCL3, anti-CCL4, and anti-CCL5), and IgG controls. Chosen chemokine concentrations were those that produced 50% maximal chemotaxis when tested individually (a pool of 3 ng/mL CCL3, 10 ng/mL CCL4, and 3 ng/mL CCL5 was used in CCR5 experiments and a mixture of 20 ng/ mL CCL3 and 5 ng/mL CCL5 was used in CCR1 experiments). doi:10.1371/journal.pone.0043332.g005

Article Snippet: Control inhibitors included vCCI-Fc (produced at VLST – the Fc is from human IgG1 with mutations to prevent interactions with Fc receptors [38]) and commercial antibodies against CCL3 (R&D Systems #MAB270), CCL4 (R&D Systems #MAB271), and CCL5 (R&D Systems #MAB278) as well as IgG controls.

Techniques: Inhibition, Chemotaxis Assay, Recombinant, Produced

Journal: PloS one

Article Title: A novel highly potent therapeutic antibody neutralizes multiple human chemokines and mimics viral immune modulation.

doi: 10.1371/journal.pone.0043332

Figure Lengend Snippet: Figure 8. SCID-hu mouse model of leukocyte migration. (A) NSG (NOD/SCID/IL2r-cnull) mice were injected i.v. with human PBMC and allowed to engraft for 10 d. MAb d5d7 was administered i.v. just before chemokines were injected s.c. in Matrigel. After 7 d the skin sites were harvested and single cell suspensions were generated. Human leukocytes were tagged with specific antibodies and analyzed by flow cytometry. (B) Inhibition by MAb d5d7 of skin leukocyte migration into chemokine-embedded Matrigel plugs in NSG mice engrafted with human PBMC. The negative control group consisted of animals treated with s.c. injection of Matrigel + PBS and i.v. administration of control IgG. All other groups had s.c. injections of Matrigel containing CCL3, CCL4, and CCL5 (400 ng each) with i.v. administration of PBS, control IgG, or MAb d5d7 antibody. Data were analyzed using a student t test. doi:10.1371/journal.pone.0043332.g008

Article Snippet: Control inhibitors included vCCI-Fc (produced at VLST – the Fc is from human IgG1 with mutations to prevent interactions with Fc receptors [38]) and commercial antibodies against CCL3 (R&D Systems #MAB270), CCL4 (R&D Systems #MAB271), and CCL5 (R&D Systems #MAB278) as well as IgG controls.

Techniques: Migration, Injection, Generated, Flow Cytometry, Inhibition, Negative Control, Control

Journal: Fertility and sterility

Article Title: Extracellular matrix signaling activates differentiation of adult ovary-derived oogonial stem cells in a species-specific manner

doi: 10.1016/j.fertnstert.2018.12.015

Figure Lengend Snippet: (A) Representative images of in vitro derived (IVD) oocytes formed by mouse oogonial stem cells (mOSCs) in culture. Scale bar 50-µm. Asterisk (upper right panel) highlights an OSC shown for size comparison to an adjacent IVD oocyte. (B) Number of IVD oocytes formed in cultures of mOSCs seeded onto tissue culture plastic (TCP), laminin, type I collagen (Col 1), type IV collagen (Col 4) or a mixture of the two collagens (Col 1 + 1 4; 10 µg/ cm2 of a 1:1 mixture) for 72 hours (mean ± SEM; n = 5; *P<.05 vs. TCP). (C) Representative expression analysis of known matrix-binding integrin subunits in freshly isolated (fresh) or passage 35 (cultured) mOSCs (+RT and –RT = addition and exclusion of reverse transcriptase for the reverse-transcription polymerase chain reaction analysis to rule out the possibility of signal resulting from amplification of genomic DNA). (D) Treatment of mOSCs with the integrin-neutralizing peptide Arg-Gly-Asp (RGD; 500 µmol/L) inhibits the increase in mOSC differentiation induced by a mixture of type I and type IV collagens (mean T SEM; n = 3; *P<.05; **P<.01). (E) Addition of RGD peptide inhibits the ability of the collagen mixture to increase expression of the meiotic commitment gene Stra8 in cultured mOSCs (n = 5; *P<.05).

Article Snippet: Quantitative analysis of stimulated by retinoic acid gene 8 ( Stra8 ) expression was performed with the use of a Taqman gene expression assay against Stra8 (assay ID Mm00486473_m1) and a housekeeping reference gene ( glyceraldehyde 3-phosphate dehydrogenase [ GAPDH ]; assay ID Mm99999915_g1).

Techniques: In Vitro, Derivative Assay, Comparison, Expressing, Binding Assay, Isolation, Cell Culture, Reverse Transcription, Polymerase Chain Reaction, Amplification

Journal: JACC: Basic to Translational Science

Article Title: Fibroblast-Specific Depletion of Human Antigen R Alleviates Myocardial Fibrosis Induced by Cardiac Stress

doi: 10.1016/j.jacbts.2024.03.004

Figure Lengend Snippet: Myofibroblast-Specific Deletion of HuR Preserves Cardiac Function in Pressure Overload–Induced Cardiac Injury (A) Study design: 12-week-old mice were fed a tamoxifen (TAM) diet for 2 weeks before transverse aortic constriction (TAC) surgery and maintained on the TAM diet until the end of the study. (B) Flow cytometric analysis of human antigen R (HuR)-positive (CD45 – CD31 – PDGFR-α + HuR + ) cardiac fibroblasts (FBs) after 8 weeks of TAC. Left panel: representative flow images for CD45 – CD31 – PDGFR-α + –gated fibroblasts. Fluorescence-tagged secondary antibody was used as a control for intracellular staining of HuR (denoted as secondary antibody control). Right panel: quantification of CD45 – CD31 – PDGFR-α + HuR + fibroblasts in control (CT) and knockout (KO) mice (CT, n = 8; KO, n = 8, ∗∗∗ P < 0.001 vs CT mice). Values are median with 25th and 75th percentiles and were analyzed with the Mann-Whitney U test. Evaluation of cardiac parameters by echocardiography: ejection fraction (EF) (∗∗∗∗ P < 0.0001 vs CT) (C) and fractional shortening (FS) (∗∗∗∗ P < 0.0001 vs CT) (D). Values are mean ± SEM (CT, n > 5; KO, n > 5) and were analyzed by using a two-way analysis of variance with Tukey post hoc analysis. PDGFR-α = platelet-derived growth factor receptor-α.

Article Snippet: In some experiments, fibroblasts were enriched by using the Magnetic cell isolation and cell separation kit (catalog no. 130-042-301; Miltenyi Biotec) with recommended dilutions of antibodies against CD31 (catalog no. 130-097-418; Miltenyi Biotec) and CD45 (catalog no. 130-052-301; Miltenyi Biotec) as per the manufacturer’s instructions.

Techniques: Fluorescence, Control, Staining, Knock-Out, MANN-WHITNEY, Derivative Assay

Journal: JACC: Basic to Translational Science

Article Title: Fibroblast-Specific Depletion of Human Antigen R Alleviates Myocardial Fibrosis Induced by Cardiac Stress

doi: 10.1016/j.jacbts.2024.03.004

Figure Lengend Snippet: Myofibroblast HuR Promotes Myocardial Fibrosis in Pressure Overload Injury (A) Assessment of cardiac fibrosis by Masson’s trichrome staining after 8 weeks of TAC surgery. Left panel: representative trichrome-stained left ventricular regions showing interstitial and perivascular fibrosis; right panel: quantification of left ventricular fibrosis (CT, n = 5; KO, n = 6; ∗ P < 0.05 vs CT). (B) Flow cytometric assessment of CD45 – CD31 – PDGFR-α + α-SMA + fibroblasts in the left ventricles of CT and KO mice. Left panel: representative flow images for CD45 – CD31 – PDGFR-α + –gated fibroblasts. Fluorescence-tagged secondary antibody was used as a control for intracellular staining of α-SMA (denoted as secondary antibody control). Right panel: quantification of CD45 – CD31 – PDGFR-α + α-SMA + fibroblasts in CT and KO mice (CT, n = 8; KO, n = 8; ∗∗ P < 0.01 vs CT). Myocardial gene expression of Collagen type I alpha 1 chain (COL1A1) (C) and Collagen type III alpha 1 chain (COL3A1) (D) in the left ventricles of CT and KO mice normalized to GAPDH (CT, n = 5; KO, n = 8; COL1A1: ∗ P < 0.05, COL3A1: P = ns vs CT). (E) Real-time quantitative polymerase chain reaction analysis of myofibroblast markers (periostin [Postn], α-SMA, and vimentin) in enriched cardiac fibroblasts of CT and KO mice after 8 weeks of TAC surgery (n = 3 mice per group per experiment; the samples were pooled and repeated twice). Values are mean ± SEM. Data in A and D were analyzed by using an unpaired Welch's t -test. Data in B and C were analyzed by using an unpaired Student’s t -test. Abbreviations as in Figure 1 , Figure 2 , Figure 3 .

Article Snippet: In some experiments, fibroblasts were enriched by using the Magnetic cell isolation and cell separation kit (catalog no. 130-042-301; Miltenyi Biotec) with recommended dilutions of antibodies against CD31 (catalog no. 130-097-418; Miltenyi Biotec) and CD45 (catalog no. 130-052-301; Miltenyi Biotec) as per the manufacturer’s instructions.

Techniques: Staining, Fluorescence, Control, Gene Expression, Real-time Polymerase Chain Reaction

Journal: JACC: Basic to Translational Science

Article Title: Fibroblast-Specific Depletion of Human Antigen R Alleviates Myocardial Fibrosis Induced by Cardiac Stress

doi: 10.1016/j.jacbts.2024.03.004

Figure Lengend Snippet: Deletion of HuR Reduces Cardiac Myofibroblast Proliferation After Injury (A) Flow cytometric analysis of CD31 – CD45 – α-SMA + BrdU + cells in the heart of the CT and KO mice after 1 week of TAC injury using 5-bromo-2′-deoxyuridine (BrdU) labeling. Left panel: representative flow cytometry images. Right panel: quantification of CD45 – CD31 – α-SMA + BrdU + cells in CT and KO mice (CT, n = 4; KO, n = 3; ∗ P < 0.05 vs CT; BrdU administration: 10 mg/kg body weight). (B) In vitro cell proliferation was measured by using the MTT assay in siControl and siHuR HCFs treated with TGF-β1 for 48 hours (n = 4 per group; ∗ P < 0.05 vs siControl). (C) Expression of Ki67 was assessed by using Western blot analysis in siControl and siHuR HCFs treated with TGF-β1 for 24 hours and normalized to β-tubulin (n > 5/group; ∗∗∗∗ P < 0.0001 vs siControl). Values are mean ± SEM and were analyzed by using an unpaired Student’s t -test. Abbreviations as in Figures 1 and .

Article Snippet: In some experiments, fibroblasts were enriched by using the Magnetic cell isolation and cell separation kit (catalog no. 130-042-301; Miltenyi Biotec) with recommended dilutions of antibodies against CD31 (catalog no. 130-097-418; Miltenyi Biotec) and CD45 (catalog no. 130-052-301; Miltenyi Biotec) as per the manufacturer’s instructions.

Techniques: Labeling, Flow Cytometry, In Vitro, MTT Assay, Expressing, Western Blot

Journal: PloS one

Article Title: Inhibition of E2F1/CDK1 pathway attenuates neuronal apoptosis in vitro and confers neuroprotection after spinal cord injury in vivo.

doi: 10.1371/journal.pone.0042129

Figure Lengend Snippet: Figure 1. Spinal cord injury induces upregulation of expression of E2F1 and its downstream targets. Analysis of expression of E2F1 and its transcriptional target genes in intact and injured spinal cord were performed by western blotting. A. 5-mm-long segment centered at the injury epicenter was dissected and homogenized in RIPA buffer. Equal amounts of protein were electrophoretically separated on NuPAGE Novex Bis-Tris gradient gels, transferred to nitrocellulose membranes, and blotted with antibodies to E2F1 and CDK1. GAPDH signal served as a loading control. B– C. The signal quantifications for E2F1 (B) and CDK1 (C) using Gel-Pro Analyzer software are displayed. E2F1 and CDK1 expression level was upregulated as early as 15 min and sustained until 3 days after injury. D–E. Cyclin A expression was increased at all time points. F. Bim and c-Myb, downstream targets of E2F1 were upregulated as early as 5 h post-injury and sustained until day 3 after SCI. G–H. Quantification of respective western blots in panel D. N = 4 rats/time point. *P,0.05 vs sham group. doi:10.1371/journal.pone.0042129.g001

Article Snippet: 27 mer siRNA duplexes for human E2F1 (ID 1869), trilencer-27 universal scrambled negative control siRNA duplex (SR30004), constructs expressing 29 mer shRNAs against Rat Cdc2 and E2F1 in pGFPV-RS vectors were obtained from Origene Technologies Inc. Rat Neuron NucleofectorH Kit (VPG-1003) was purchased from Lonza.

Techniques: Expressing, Western Blot, Control, Software

Journal: PloS one

Article Title: Inhibition of E2F1/CDK1 pathway attenuates neuronal apoptosis in vitro and confers neuroprotection after spinal cord injury in vivo.

doi: 10.1371/journal.pone.0042129

Figure Lengend Snippet: Figure 2. CDK1 activity is increased after spinal cord injury. Western blotting analysis of common CDK substrates, CDK1 co-activator cyclin B1 and phosphorylations of specific CDK1 substrate (Ser54)-n-myc was performed in homogenates obtained from intact and injured spinal cord. A. Cyclin B1 expression was upregulated at all time points tested. Phosphorylation (Ser54) of n-myc and phospho-CDK substrate motif signal levels were increased from 5 h to day 7. B–D. Quantification of respective western blots in panel A. n = 4 rats/time point. *P,0.05 vs sham group. doi:10.1371/journal.pone.0042129.g002

Article Snippet: 27 mer siRNA duplexes for human E2F1 (ID 1869), trilencer-27 universal scrambled negative control siRNA duplex (SR30004), constructs expressing 29 mer shRNAs against Rat Cdc2 and E2F1 in pGFPV-RS vectors were obtained from Origene Technologies Inc. Rat Neuron NucleofectorH Kit (VPG-1003) was purchased from Lonza.

Techniques: Activity Assay, Western Blot, Expressing, Phospho-proteomics

Journal: PloS one

Article Title: Inhibition of E2F1/CDK1 pathway attenuates neuronal apoptosis in vitro and confers neuroprotection after spinal cord injury in vivo.

doi: 10.1371/journal.pone.0042129

Figure Lengend Snippet: Figure 3. The temporal profile and cell specificity of E2F1 and CDK1 expression after SCI. A–B. Coronal section in intact spinal cord (A) showed that E2F1 is relatively weak and detected mainly in neurons of the gray matter. At 24 h after SCI, E2F1 immunoreactivity was upregulated not only in gray matter but also in lesion area (B). C. E2F1+ cells were also co-labelled with NeuN in the dorsal horn of the gray matter at 1 day after SCI. D–E. Only a small subset of E2F1+ cells in the lesion area were positive for OX42 at 24 h (D) and 7 d (E) after SCI. F–G. In the intact spinal cord (F), CDK1 immunoreactivity is relatively weak and detected mainly in motor neurons in the ventral horn and CC1+ oligodendrocytes. At 24 h after SCI (G), CDK1 immunoreactivity was upregulated not only in the ventral horn but also in the spared white matter, colocalized with CC1+ oligodendrocytes. CDK1+ cells also appeared in the lesion area. H–I. CDK1 was expressed by CC1+ oligodendrocytes in the white matter in the intact spinal cord (H) and at 1 day after SCI. J. Only a small subset of CDK1+ cells in the lesion area were positive for OX42 at 24 h after SCI. K. Coronal section in intact spinal cord (a) shows that E2F1 was expressed in the motor neurons in the ventral horn. Immunoreactivity of E2F1 (b–d) was increased at 5 h, and 1–3 days post injury, and highly expressed by motor neurons. L. In intact spinal cord (a), CDK1/NeuN was detected in the motor neurons in the ventral horn. At 5 h after injury, immunoreactivity of CDK1 (b) was increased and sustained until 3 days post injury (c–d), and highly expressed by motor neurons. All images were taken at 2 mm rostral to epicenter. Scale bar = 500 mm for A–B, F–G. Scale bar = 100 mm for C–E, H–J, K–L. doi:10.1371/journal.pone.0042129.g003

Article Snippet: 27 mer siRNA duplexes for human E2F1 (ID 1869), trilencer-27 universal scrambled negative control siRNA duplex (SR30004), constructs expressing 29 mer shRNAs against Rat Cdc2 and E2F1 in pGFPV-RS vectors were obtained from Origene Technologies Inc. Rat Neuron NucleofectorH Kit (VPG-1003) was purchased from Lonza.

Techniques: Expressing

Journal: PloS one

Article Title: Inhibition of E2F1/CDK1 pathway attenuates neuronal apoptosis in vitro and confers neuroprotection after spinal cord injury in vivo.

doi: 10.1371/journal.pone.0042129

Figure Lengend Snippet: Figure 4. E2F1 gene silencing down-regulates endogenous CDK1 expression in vitro. A. 27 mer siRNA duplexes for human E2F1 or trilencer-27 universal scrambled negative control siRNA duplex was transfected in the human neuroblastoma SH-SY5Y cells. Two days after transfection, the cells were harvested and subjected to western blotting using mouse monoclonal antibodies to E2F1 and CDK1. Transfection with shRNA against E2F1 resulted in reduction of E2F1 expression (58% to 66% of control), accompanied by 50% of reduction of CDK1 expression. B. Primary rat cerebral cortical neurons were transfected with shRNA against rat E2F1. E2F1 protein expression was robust reduced to 47% or 59% for shRNAs 1 and 2 respectively, and E2F1 knockdown resulted in reduction of CDK1 expression from 47% to 64% for shRNAs 1 and 2 respectively. N = 4 dishes from 3 independent culture. *P,0.05 vs sham group. doi:10.1371/journal.pone.0042129.g004

Article Snippet: 27 mer siRNA duplexes for human E2F1 (ID 1869), trilencer-27 universal scrambled negative control siRNA duplex (SR30004), constructs expressing 29 mer shRNAs against Rat Cdc2 and E2F1 in pGFPV-RS vectors were obtained from Origene Technologies Inc. Rat Neuron NucleofectorH Kit (VPG-1003) was purchased from Lonza.

Techniques: Expressing, In Vitro, Negative Control, Transfection, Western Blot, Bioprocessing, shRNA, Control, Knockdown

Journal: PloS one

Article Title: Inhibition of E2F1/CDK1 pathway attenuates neuronal apoptosis in vitro and confers neuroprotection after spinal cord injury in vivo.

doi: 10.1371/journal.pone.0042129

Figure Lengend Snippet: Figure 5. Colocalization of E2F1/CDK1 upregulation with neuronal apoptosis in injured spinal cord. A–C. Western blot analysis shows a significant increase in biochemical markers of apoptosis, active caspase-3 signal, as well as 145/150 kDa cleavage product of a-fodrin after SCI. N = 4 rats/time points. *p,0.05 vs sham group. D. E2F1+ cells were co-label with cleaved caspase 3 (yellow, arrow heads) in the gray matter at 2 mm rostral to the epicenter at 1 day after SCI. Scale bar = 100 mm. E. Coronal section in intact spinal cord (top panel) shows that CDK1 was expressed in the motor neurons in the ventral horn (VH). At 1 day after injury, immunoreactivity of CDK1 (middle panel, green) was increased, and highly expressed by apoptotic motor neurons (red), as shown at 2 mm rostral to epicenter. CDK1 was rarely expressed by inter-neurons in the dorsal horn (DH) after SCI (bottom panel). Scale bar = 100 mm for D(a–h) and 500 mm for D(i–l). doi:10.1371/journal.pone.0042129.g005

Article Snippet: 27 mer siRNA duplexes for human E2F1 (ID 1869), trilencer-27 universal scrambled negative control siRNA duplex (SR30004), constructs expressing 29 mer shRNAs against Rat Cdc2 and E2F1 in pGFPV-RS vectors were obtained from Origene Technologies Inc. Rat Neuron NucleofectorH Kit (VPG-1003) was purchased from Lonza.

Techniques: Western Blot

Journal: PloS one

Article Title: Inhibition of E2F1/CDK1 pathway attenuates neuronal apoptosis in vitro and confers neuroprotection after spinal cord injury in vivo.

doi: 10.1371/journal.pone.0042129

Figure Lengend Snippet: Figure 6. E2F1/CDK1 expression is necessary for trophic deprivation-induced neuronal apoptosis. Rat cortical neurons were co- transfected with expression plasmids for ß-galactosidase with, either empty vector or vector expressing E2F1 or CDK1 (A and B). Similarly, ß- galactosidase plasmid was co-transfected along with scrambled, E2F1 or CDK1 shRNAs (C, D and E) and the extent of apoptosis was examined 48 h after transfection, or after an additional 24 h of trophic deprivation (TD) induction post 48 h transfection. A. Neurons transfected with E2F1 vector (0.6 mg DNA/0.56106 neurons) increased basal apoptosis as compared to empty vector. B. Neurons transfected with CDK1 (0.8 mg DNA/0.56106

Article Snippet: 27 mer siRNA duplexes for human E2F1 (ID 1869), trilencer-27 universal scrambled negative control siRNA duplex (SR30004), constructs expressing 29 mer shRNAs against Rat Cdc2 and E2F1 in pGFPV-RS vectors were obtained from Origene Technologies Inc. Rat Neuron NucleofectorH Kit (VPG-1003) was purchased from Lonza.

Techniques: Expressing, Transfection, Plasmid Preparation

Journal: PloS one

Article Title: Inhibition of E2F1/CDK1 pathway attenuates neuronal apoptosis in vitro and confers neuroprotection after spinal cord injury in vivo.

doi: 10.1371/journal.pone.0042129

Figure Lengend Snippet: Figure 7. Pharmacological inhibition of CDK1 blocks neuronal apoptosis. Cortical neurons were pre-treated with Roscovitine or CR8 (CDK1 inhibitors) or vehicle and then exposed to TD-or campthotecin induced apoptosis. A. Representative photomicrographs of control and trophic deprived neurons treated with the indicated concentrations Roscovitine and CR8 are shown. Upper row presents phase contrast images (Healthy neurons are indicated by larger cell bodies and abundant processes; Apoptotic neurons display shrunken cell bodies and sparse or lost processes). Lower row shows chromatin staining with Hoechst 33258. Arrows and arrowheads indicate surviving and apoptotic neurons, respectively suggesting an attenuation of TD-induced neuronal death in neurons pre-treated with Roscovinine or CR8. B. A quantitative assessment of the percentage of nuclei featuring chromatin condensation demonstrates a significant attenuation of TD-induced apoptosis in neurons pre-treated with Roscovitine (10 mM; *p,0.05, vs. TD vehicle) whereas CR8 at concentrations as low as 1 mM (***p,0.001, vs. TD vehicle) almost completely blocked development of apoptotic features in neuronal nuclei. C. Significant attenuation of campthotecin-induced apoptosis in neurons pre-treated with Roscovitine (50 mM; *p,0.001, vs. vehicle) and CR8 at concentrations as low as 1 mM (***p,0.001, vs. vehicle). doi:10.1371/journal.pone.0042129.g007

Article Snippet: 27 mer siRNA duplexes for human E2F1 (ID 1869), trilencer-27 universal scrambled negative control siRNA duplex (SR30004), constructs expressing 29 mer shRNAs against Rat Cdc2 and E2F1 in pGFPV-RS vectors were obtained from Origene Technologies Inc. Rat Neuron NucleofectorH Kit (VPG-1003) was purchased from Lonza.

Techniques: Inhibition, Control, Staining

Journal: PloS one

Article Title: Inhibition of E2F1/CDK1 pathway attenuates neuronal apoptosis in vitro and confers neuroprotection after spinal cord injury in vivo.

doi: 10.1371/journal.pone.0042129

Figure Lengend Snippet: Figure 8. CR8 administration reduces SCI-induced activation of the E2F1/CDK1 signaling pathway. Samples were obtained from rats exposed to spinal cord injury and CR8 treatment (1 mg/kg intraperitoneal administration) and analyzed by western blotting. Equal protein loading is demonstrated by consistent GAPDH levels. A. CR8 attenuated SCI mediated increase in E2F1 and its target cyclin A expression. B–C. Quantification of respective western blots in panel A. D. CR8 reduced SCI induced increase in phospho-(Ser54)-n-myc, phosphorylated CDK substrates and expression of cyclin B1. E–G. Quantification of respective western blots in panel E. H–J. Administration of CR8 significantly reduced Bim and c-Myb expression at 24 h after SCI. H shows representative Western blots for Bim, c-Myb, and the loading control, GAPDH. I and J show quantitative analysis of Bim and c- Myb expression. N = 4. *p,0.05 vs. vehicle group. doi:10.1371/journal.pone.0042129.g008

Article Snippet: 27 mer siRNA duplexes for human E2F1 (ID 1869), trilencer-27 universal scrambled negative control siRNA duplex (SR30004), constructs expressing 29 mer shRNAs against Rat Cdc2 and E2F1 in pGFPV-RS vectors were obtained from Origene Technologies Inc. Rat Neuron NucleofectorH Kit (VPG-1003) was purchased from Lonza.

Techniques: Activation Assay, Western Blot, Expressing, Control

Journal: PloS one

Article Title: Inhibition of E2F1/CDK1 pathway attenuates neuronal apoptosis in vitro and confers neuroprotection after spinal cord injury in vivo.

doi: 10.1371/journal.pone.0042129

Figure Lengend Snippet: Figure 9. SCI-induced immunoreactivity of E2F1 and CDK1 was attenuated by CR8 treatment. A. Coronal section in intact spinal cord (a– c) shows that E2F1 was expressed in the motor neurons in the ventral horn. At 1 day after injury, immunoreactivity of E2F1 (d–f) was increased, and highly expressed by motor neurons. The upregulation of E2F1 was clearly attenuated by CR8 treatment (g–i). B. In intact spinal cord (a–c), CDK1/ NeuN was detected in the motor neurons in the ventral horn. At 1 day after injury, immunoreactivity of CDK1 (d–f) was increased, and highly expressed by motor neurons. CDK1 upregulation was attenuated by CR8 treatment (g–i). All images were taken at 2 mm rostral to epicenter. Scale bar = 100 mm for C–F. doi:10.1371/journal.pone.0042129.g009

Article Snippet: 27 mer siRNA duplexes for human E2F1 (ID 1869), trilencer-27 universal scrambled negative control siRNA duplex (SR30004), constructs expressing 29 mer shRNAs against Rat Cdc2 and E2F1 in pGFPV-RS vectors were obtained from Origene Technologies Inc. Rat Neuron NucleofectorH Kit (VPG-1003) was purchased from Lonza.

Techniques:

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Long-Term Impact of Toxoplasma gondii Infection on Human Monocytes

doi: 10.3389/fcimb.2019.00235

Figure Lengend Snippet: Proportion of CD62L + , CD64 + , CCR2 + , HLA,A,B,C + or HLA,DR,DP,DQ + cells among peripheral blood monocytes from donors with or without chronic toxoplasmosis. Plastic-adherent PBMCs were isolated from blood samples and were fluorescently labeled using FITC-conjugated anti-CD14 or antibodies directed against the indicated surface markers or isotype control antibodies and PE-conjugated secondary antibodies. Blood donors were serologically classified as chronically T. gondii -infected or non-infected using plasma from the blood samples. (A) CD14 + monocytes (R1) were back-gated and identified (R2) among FSC/SSC-analyzed total cells. R2-gated cells were then analyzed for expression of cell surface markers as indicated, and positive cells were identified after specific (anti-CCR2 in (A) ; see for the other surface markers) and isotype control labeling. (B–F) Percentages of monocytes from T. gondii seropositive or seronegative individuals positive for cell surface markers as indicated. Solid and dashed lines in the box-whisker plots indicate median and mean values, respectively; circles indicate individual data points. Data are from 5 T. gondii seropositive and 16 seronegative blood donors; outlyers were excluded. * p < 0.05 (Student's t -test).

Article Snippet: Alternatively, they were labeled with 2 μg/mL of mouse monoclonal antibodies directed against human CD62L (clone DREG-56), human CD64 (clone 10.01.13), human HLA-A,B,C (clone G46-2.6), human HLA-DR,DP,DQ (clone Tu39; all antibodies from BD Biosciences), or directed against human CCR2 (R&D Systems, Wiesbaden-Nordenstadt, Germany), or they were incubated with appropriate isotype control antibodies (clones MOPC-21, 27-35 and G155-178; BD Biosciences) for 30 min at 4°C.

Techniques: Isolation, Labeling, Control, Infection, Clinical Proteomics, Expressing, Whisker Assay

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Long-Term Impact of Toxoplasma gondii Infection on Human Monocytes

doi: 10.3389/fcimb.2019.00235

Figure Lengend Snippet: Expression of cell surface receptors on monocytes from individuals with chronic toxoplasmosis and non-infected controls.

Article Snippet: Alternatively, they were labeled with 2 μg/mL of mouse monoclonal antibodies directed against human CD62L (clone DREG-56), human CD64 (clone 10.01.13), human HLA-A,B,C (clone G46-2.6), human HLA-DR,DP,DQ (clone Tu39; all antibodies from BD Biosciences), or directed against human CCR2 (R&D Systems, Wiesbaden-Nordenstadt, Germany), or they were incubated with appropriate isotype control antibodies (clones MOPC-21, 27-35 and G155-178; BD Biosciences) for 30 min at 4°C.

Techniques: Expressing

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Long-Term Impact of Toxoplasma gondii Infection on Human Monocytes

doi: 10.3389/fcimb.2019.00235

Figure Lengend Snippet: Impact of in vitro infection of monocyte-enriched PBMCs with T. gondii on expression of CD62L, CD64, CCR2, HLA-A,B,C, or HLA-DR,DP,DQ on monocytes from donors with or without chronic toxoplasmosis. Monocyte-enriched PBMCs were isolated from blood samples and were either directly FACS-analyzed (0 h) or were cultivated in vitro for 48 h and infected or not with T. gondii during the final 24 h as indicated and then FACS-analyzed. Expression of cell surface markers was determined for CD14-positive monocytes as outlined in . (A–E) Percentages of cells from T. gondii seropositive (gray bars) or seronegative (open bars) individuals with expression of surface markers above background staining. (F–J) Expression levels of surface markers as indicated on monocytes from T. gondii seropositive or seronegative blood donors. Data represent means ± S.E.M. from 5 T. gondii seropositive and from 13 out of 16 seronegative blood donors which had been randomly selected for in vitro infection assays; outlyers were excluded. Individual data points are also indicated. Significant differences between groups were identified by ANOVA [ *** p < 0.001; ** p < 0.01; * p < 0.05; a and b indicate dose-dependent increase or decrease, respectively, of HLA-DR,DP,DQ after parasite infection ( p < 0.01)].

Article Snippet: Alternatively, they were labeled with 2 μg/mL of mouse monoclonal antibodies directed against human CD62L (clone DREG-56), human CD64 (clone 10.01.13), human HLA-A,B,C (clone G46-2.6), human HLA-DR,DP,DQ (clone Tu39; all antibodies from BD Biosciences), or directed against human CCR2 (R&D Systems, Wiesbaden-Nordenstadt, Germany), or they were incubated with appropriate isotype control antibodies (clones MOPC-21, 27-35 and G155-178; BD Biosciences) for 30 min at 4°C.

Techniques: In Vitro, Infection, Expressing, Isolation, Staining

Journal: Journal of Biological Chemistry

Article Title: Transcriptional Regulation of the Bovine Leukemia Virus Promoter by the Cyclic AMP-response Element Modulator τ Isoform

doi: 10.1074/jbc.m703060200

Figure Lengend Snippet: FIGURE2.A,characterizationoftheproteincomplexesontheBLVTxRE1.Analysesoftheproteincompositionofthe threeretardedcomplexesobtainedbyincubationoftheTxRE1probewithYR2nuclearextractswereperformedby supershiftassays.PriortotheadditionoftheTxRE1probetothebindingreaction,nuclearextractsfromPI-treated BLV-infected YR2 cells were incubated with purified rabbit IgG as negative control (lane 1), with the anti-CREB antibody(lane2),theanti-CREMantibody(lane3),theanti-ATF-1antibody(lane4),theanti-ATF-2antibody(lane5), or with different combinations of these antibodies (lanes 6–11). The probe was then added, and the binding reac- tionswereanalyzedbyPAGE.Theretardedcomplexeswerevisualizedbyautoradiography.ThemajorDNA-protein complexes C1, C2, and C3 are indicated byarrows, and the supershifted (S-CREB, S-CREM, S-ATF-1, S-ATF-2) or super- supershifted (SS-CREB CREM, SS-CREMATF-1, SS-CREBATF-1) complexes are indicated by numbers. The CRE- mutatedTxRE1probewasusedascontrolinordertodemonstratethatthebindingofCREB,CREM,ATF-1,andATF-2 to the TxRE1 probe required intact viral CRE motifs (lanes 12–16). B, as controls for cross-reactivity between the anti-CREM antibody and CREB proteins and between the anti-CREB antibody and CREM proteins, we performed Westernblot(WB)analysisusinginvitrotranslatedCREBandCREMproteinsandeithertheanti-CREMantibody(top panel) or the anti-CREB antibody (bottom panel). The in vitro translated proteins were generated by using the TNT CoupledWheatGermExtractSystem(Promega)withthepSV-CREM-wtexpressionvectorencodingmurineCREM (TNT-CREM, lane 1), the pSG-CREB2 expression plasmid encoding bovine CREB2 (TNT-CREB, lane 3), and the pSG5 empty vector as control (TNT control, lane 2). C, CREM proteins are expressed in uninfected ovine PBMCs. Supershift experiments were performed with nuclear extracts of PBMCs isolated from a healthy sheep. Prior to the addition of theTxRE1probetothebindingreaction,thePBMCnuclearextractswereincubatedwitheitherapurifiedrabbitIgG asnegativecontrol(lane1)ortheanti-CREMantibody(lane2).Theprobewasthenadded,andthebindingreactions wereanalyzedbyPAGE.Theretardedcomplexeswerevisualizedbyautoradiography.TheDNA-proteincomplexes N1 and N2 and the supershifted complex S-CREM are indicated by arrows.

Article Snippet: For supershift assays, monoclonal antibodies against ATF-1 (Santa Cruz Biotechnology, catalog number sc-243X) andATF-2 (catalog number sc-242X) and polyclonal antibodies against human CREB (catalog number sc-240X) and human full-length CREM (catalog number sc-440X) were added at a final concentration of 2 g/reaction mixture at the beginning of the binding reaction for 20 min before adding the DNA probe (blocking conditions).

Techniques: Infection, Incubation, Purification, Negative Control, Binding Assay, In Vitro, Generated, Expressing, Plasmid Preparation, Control, Isolation

Journal: Journal of Biological Chemistry

Article Title: Transcriptional Regulation of the Bovine Leukemia Virus Promoter by the Cyclic AMP-response Element Modulator τ Isoform

doi: 10.1074/jbc.m703060200

Figure Lengend Snippet: FIGURE 3. Recruitment of CREB, CREM, ATF-1, and ATF-2 to the TxRE pro- moter region in vivo. ChIP assays were used to detect binding of bZIP tran- scription factors to the TxRE region of the BLV promoter in the chromosomal context of proviruses integrated in YR2 cells. DNA and protein were cross- linked with formaldehyde for 10 min, and DNA was sheared. The cross-linked protein-DNA complexes were immunoprecipitated (IP) with the anti-CREB antibody (lane 4), the anti-ATF-1 antibody (lane 5), the anti-ATF-2 antibody (lane 6), the anti-CREM antibody (lane 7), or with a purified rabbit IgG as neg- ative control (lane 3). The protein-DNA cross-links were reversed, and the purified DNAs were amplified by semi-quantitative radioactive PCR using a primer set amplifying the BLV promoter TxRE region (nt 172 to 29). PCR of the input (sample representing PCR amplification from a 1:25 dilution of total input chromatin from the ChIP experiment) is shown in lane 2. The PCR con- trol represents the PCR amplification in the absence of DNA (lane 1).

Article Snippet: For supershift assays, monoclonal antibodies against ATF-1 (Santa Cruz Biotechnology, catalog number sc-243X) andATF-2 (catalog number sc-242X) and polyclonal antibodies against human CREB (catalog number sc-240X) and human full-length CREM (catalog number sc-440X) were added at a final concentration of 2 g/reaction mixture at the beginning of the binding reaction for 20 min before adding the DNA probe (blocking conditions).

Techniques: In Vivo, Binding Assay, Immunoprecipitation, Purification, Control, Amplification