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 Veterinary Diagnostic Investigation : Official Publication of the American Association of Veterinary Laboratory Diagnosticians, Inc

Article Title: Activated platelets and platelet-leukocyte aggregates in the equine systemic inflammatory response syndrome

doi: 10.1177/10406387221077969

Figure Lengend Snippet: Percentages of activated platelets (CD62P-positive) and platelet-leukocyte aggregates (PLAs; CD11a/18-positive) in platelet-leukocyte-rich plasma (PLRP) and after activation with collagen (50 μg/mL) measured with Accuri C6 (Becton Dickinson) and the BD Accuri C6 analysis software in 10 healthy adult horses (median, IQR; Prism v.6, GraphPad; Wilcoxon test).

Article Snippet: Activation of platelets was determined with fluorescein isothiocyanate (FITC)-conjugated (LYNX rapid antibody conjugation kit; Bio-Rad) monoclonal mouse anti-human antibodies against CD62P (MCA2419, dilution 1:50 with modified HEPES/Tyrod buffer).

Techniques: Activation Assay, Software

Journal: Journal of Veterinary Diagnostic Investigation : Official Publication of the American Association of Veterinary Laboratory Diagnosticians, Inc

Article Title: Activated platelets and platelet-leukocyte aggregates in the equine systemic inflammatory response syndrome

doi: 10.1177/10406387221077969

Figure Lengend Snippet: Platelet activation and platelet-leukocyte aggregates (PLAs) in controls ( n = 10) and systemic inflammatory response syndrome (SIRS) group ( n = 17) in native samples (white boxes) and after in vitro activation with collagen (cross-hatched boxes); boxes are median and IQR, whiskers minimum and maximum. A. Percentage of CD62P-positive platelets; a, p = 0.0007; b, p < 0.0001. B. Percentage of PLAs: c, p = 0.048; d, p = 0.0009; e, p = 0.036.

Article Snippet: Activation of platelets was determined with fluorescein isothiocyanate (FITC)-conjugated (LYNX rapid antibody conjugation kit; Bio-Rad) monoclonal mouse anti-human antibodies against CD62P (MCA2419, dilution 1:50 with modified HEPES/Tyrod buffer).

Techniques: Activation Assay, In Vitro

Journal: Journal of Veterinary Diagnostic Investigation : Official Publication of the American Association of Veterinary Laboratory Diagnosticians, Inc

Article Title: Activated platelets and platelet-leukocyte aggregates in the equine systemic inflammatory response syndrome

doi: 10.1177/10406387221077969

Figure Lengend Snippet: Platelet activation and platelet-leukocyte aggregates (PLAs) in the systemic inflammatory response syndrome (SIRS) group ( n = 13) depending on outcome in native samples (open circles) and after in vitro activation with collagen (black dots); bar = median. A. Individual percentage of CD62P-positive platelets; a, p = 0.03. B. Individual percentage of PLAs.

Article Snippet: Activation of platelets was determined with fluorescein isothiocyanate (FITC)-conjugated (LYNX rapid antibody conjugation kit; Bio-Rad) monoclonal mouse anti-human antibodies against CD62P (MCA2419, dilution 1:50 with modified HEPES/Tyrod buffer).

Techniques: Activation Assay, In Vitro

Journal: The FASEB Journal

Article Title: Timely expression and activation of YAP1 in granulosa cells is essential for ovarian follicle development

doi: 10.1096/fj.201900179RR

Figure Lengend Snippet: Expression and localization of Yap1 protein in mouse ovarian cells. A–C) Fluorescent IHC and confocal microscopy showing expression and localization of Yap1 (A), Ki67 (B), and 3β-Hsd (C) in ovarian cells using series sections of mouse ovarian tissues. The green color in A–C represents expression and localization of Yap1, Ki67, and 3β-HSD in mouse ovarian cells, respectively. A1–C1) Corresponding high-resolution images. Actin filaments were stained with rhodamine-phalloidin. Nuclei were stained with DAPI. GC, granulosa cell; LC, luteal cell; P2, secondary follicle. Scale bars, 100 μm.

Article Snippet: The antibody against Ki67 (marker of proliferation Ki-67) was from Abcam (Cambridge, United Kingdom).

Techniques: Expressing, Confocal Microscopy, Staining

Journal: The FASEB Journal

Article Title: Timely expression and activation of YAP1 in granulosa cells is essential for ovarian follicle development

doi: 10.1096/fj.201900179RR

Figure Lengend Snippet: Pharmacological inhibition of Yap1 disrupts ovarian follicle development in vitro and in vivo. A) Representative images showing the morphology of postnatal d 12 mouse ovaries incubated for 7 d in the presence or absence of VP (Verteporfin, a selective YAP1 antagonist). Scale bar, 500 μm. B) Hematoxylin and eosin (H&E) stain showing histology of control (CTL) and VP-treated ovaries. Scale bar, 50 μm. The insert image shows the histology of ovaries in postnatal d 12 mice. Please note that only early stage growing follicles (<5 layers of GCs) are present in these ovaries. C, D) Representative IHC images showing expression of Ki67 and cleaved Caspase-3 (Clvd-Cas3) in serial sections of cultured CTL and VP-treated ovarian tissues. Scale bars, 50 μm. E) Representative images showing expression of Yap1, Yap1-targeting genes [connective tissue growth factor (Ctgf), Myc, and Areg], and 3β-Hsd in the CTL and VP-treated ovaries determined by RT-PCR and Western blot. F) TUNEL assay showing the apoptosis of granulosa cells and atresia of follicles in CTL and VP-treated ovarian tissues. Scale bar, 500 μm. The percentages of TUNEL-positive follicles in CTL and VP-treated groups are also presented in a graph next to images. ***P < 0.001 compared with CTL. G) Inhibition of Yap1 decreases the number of corpora lutea in the mouse ovary. 3β-Hsd staining was used to identify the corpus luteum (CL). Scale bar, 500 μm. *P < 0.05 compared with CTL.

Article Snippet: The antibody against Ki67 (marker of proliferation Ki-67) was from Abcam (Cambridge, United Kingdom).

Techniques: Inhibition, In Vitro, In Vivo, Incubation, Staining, Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Western Blot, TUNEL Assay

Journal: Stem Cells (Dayton, Ohio)

Article Title: Follistatin‐Like 3 Enhances the Function of Endothelial Cells Derived from Pluripotent Stem Cells by Facilitating β‐Catenin Nuclear Translocation Through Inhibition of Glycogen Synthase Kinase‐3β Activity

doi: 10.1002/stem.2820

Figure Lengend Snippet: Induced pluripotent stem cell differentiation toward endothelial cells. Human iPS Cells were differentiated using StemPro serum free media supplemented with BMP4, Activin A, fibroblast growth factor (FGF), and vascular endothelial growth factor (VEGF) for 5 days. The differentiated cells were seeded on collagen IV, while CD144 positive cells were magnetically sorted on day 6 using MicroBeads Kit (Miltenyi BIotec) and culturing in EGM‐2 media (LONZA). (A) : Images show morphology of iPS Cells (left panel) and of their differentiated EC counterparts (right panel) Scale bar: 50 μm. (B) : Real time polymerase chain reaction (PCR) data revealed significantly increased early and (C) late EC marker mRNA expression (Data is means ±SEM [ n = 3], *, p < .05; **, p < .01; ***, p < .001). (D) : Western blots showing protein expression of the EC‐specific marker CD144 in iPS‐ECs and (E) corresponding densitometry. (F) : Immunofluorescent confocal images showing that iPS‐ECs express typical EC markers, CD144, KDR, CD31, and vWF, also costained with DAPI (4′,6‐diamidino‐2‐phenylindole, dihydrochloride). (G) : iPS‐ECs stained positive for CD144, were able to uptake LDL and form tubes in vitro. Scale bar: 50 μm. The data presented are representative or means (±SEM) of three independent experiments. Abbreviations: DAPI, 4′,6‐diamidino‐2‐phenylindole; EC, endothelial cell; GAPDH, Glyceraldehyde 3‐phosphate dehydrogenase; iPS, induced pluripotent stem; LDL, low density lipoprotein.

Article Snippet: Antibodies against VE‐Cadherin (CD144) (ab33168 and STJ96234), VEGFR (ab9530), GAPDH (ab8245), OCT‐4 (ab19857 and STJ72238), KLF4 (ab72543) eNOS (ab76198), and FSTL3 (STJ112317) were purchased from Abcam, UK or St John's Laboratory, UK.

Techniques: Cell Differentiation, Real-time Polymerase Chain Reaction, Marker, Expressing, Western Blot, Staining, In Vitro

Journal: Stem Cells (Dayton, Ohio)

Article Title: Follistatin‐Like 3 Enhances the Function of Endothelial Cells Derived from Pluripotent Stem Cells by Facilitating β‐Catenin Nuclear Translocation Through Inhibition of Glycogen Synthase Kinase‐3β Activity

doi: 10.1002/stem.2820

Figure Lengend Snippet: FSTL3 is highly expressed in iPS‐ECs. (A) : FSTL3 was progressively induced during EC differentiation in iPS‐ECs at the mRNA level, both preselection (pre‐iPS‐ECs) and postselection with CD144 (iPS‐ECs) (Data is means ± SEM [ n = 3], *, p < .05). (B) : Western Blot showing FSTL3 protein being expressed in parallel with the EC‐specific marker CD144 and (C) corresponding densitometry. (D) : FSTL3 secretion was progressively induced in cell culture media, as shown by ELISA (Data is means ± SEM [ n = 3], *, p < .05). (E) : Immunofluorescent images of FSTL3 in iPS‐ECs, Scale bar: 50 µm. (F) : Immunofluorescent staining showing that FSTL3 is expressed in parallel with the EC‐specific marker CD144 in iPS‐ECs. Scale bar: 50 µm. The data presented are representative or means (±SEM) of three independent experiments. Abbreviations: DAPI, 4′,6‐diamidino‐2‐phenylindole; ECs, endothelial cells; FSTL3, follistatin‐like 3; iPS, induced pluripotent stem.

Article Snippet: Antibodies against VE‐Cadherin (CD144) (ab33168 and STJ96234), VEGFR (ab9530), GAPDH (ab8245), OCT‐4 (ab19857 and STJ72238), KLF4 (ab72543) eNOS (ab76198), and FSTL3 (STJ112317) were purchased from Abcam, UK or St John's Laboratory, UK.

Techniques: Western Blot, Marker, Cell Culture, Enzyme-linked Immunosorbent Assay, Staining

Journal: Stem Cells (Dayton, Ohio)

Article Title: Follistatin‐Like 3 Enhances the Function of Endothelial Cells Derived from Pluripotent Stem Cells by Facilitating β‐Catenin Nuclear Translocation Through Inhibition of Glycogen Synthase Kinase‐3β Activity

doi: 10.1002/stem.2820

Figure Lengend Snippet: FSTL3 regulates endothelial cell (EC) marker expression in induced pluripotent stem (iPS)‐ECs. iPS‐ECs were transfected with the EX‐FSTL3 plasmid carrying the red mCherry fluorophore, or an empty control vector plasmid (EX‐mCherry). Forty‐eight hours later, FSTL3 overexpression induced (A) mRNA and (B, C) protein expression of EC markers CD144, eNOS, and KDR (Data is means ± SEM [ n = 3], **, p < .01; ***, p < .001). FSTL3 knockdown by short hairpin RNA (shRNA) in iPS‐ECs resulted in suppression of EC markers at the (D) mRNA and (E, F) protein levels when the cells were harvested 72 hours later (Data is means ± SEM [ n = 3], ***, p < .001). (G) : Fluorescent microscope images of iPS‐ECs transfected with EX‐FSTL3 plasmid carrying the red mCherry fluorophore, or an empty control vector plasmid (EX‐mCherry). Scale bar: 50 µm. (H) : Treatment of the cells with 25–50 ng/ml of recombinant FSTL3 (R FSTL3) increased EC marker expression such as CD144, eNOS, and KDR compared to phosphate buffered saline (PBS) control (R CTL) (Data is means ± SEM [ n = 3], ***, p < .001). ELISA showing FSTL3 concentration in cell culture media after FSTL3 overexpression compared to control (Data is means ± SEM [ n = 3], **, p < .01). The data presented are representative or means (±SEM) of three independent experiments. Abbreviations: FSTL3, follistatin‐like 3; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase. (I) Enzyme‐linked immunosorbent assay (ELISA) showed increased concentration of FSTL3 in the media after overexpression with EX‐FSTL3 plasmid (Data is means ± SEM [n < 3], **, p < .01

Article Snippet: Antibodies against VE‐Cadherin (CD144) (ab33168 and STJ96234), VEGFR (ab9530), GAPDH (ab8245), OCT‐4 (ab19857 and STJ72238), KLF4 (ab72543) eNOS (ab76198), and FSTL3 (STJ112317) were purchased from Abcam, UK or St John's Laboratory, UK.

Techniques: Marker, Expressing, Transfection, Plasmid Preparation, Control, Over Expression, Knockdown, shRNA, Microscopy, Recombinant, Saline, Enzyme-linked Immunosorbent Assay, Concentration Assay, Cell Culture

Journal: Stem Cells (Dayton, Ohio)

Article Title: Follistatin‐Like 3 Enhances the Function of Endothelial Cells Derived from Pluripotent Stem Cells by Facilitating β‐Catenin Nuclear Translocation Through Inhibition of Glycogen Synthase Kinase‐3β Activity

doi: 10.1002/stem.2820

Figure Lengend Snippet: FSTL3 induced vascular tube formation in vivo and significantly improved angiogenesis, neovascularization, and blood flow recovery in the hind limb ischemic model. Human induced pluripotent stem (iPS)‐ECs overexpressing FSTL3 enhanced the formation of vascular‐like tubes in vivo. (A) : iPS‐ECs overexpressing FSTL3 (EX‐FSTL3), significantly induced angiogenesis, after subcutaneous injection in severe combined immunodeficiency (SCID) mice, in comparison to the control iPS‐ECs (PBS CTL) or control iPS‐ECs expressing mCherry (EX‐mCherry), as shown by H&E staining and (B) capillary density quantification (Data is means ± SEM [ n = 3], **, p < .01). Scale bar: 50 µm. (C) : phosphate buffered saline (PBS)‐CTL and iPS‐ECs overexpressing mCherry (EX‐mCherry) or FSTL3 (EX‐FSTL3) were injected intramuscularly in NOD.CB17‐Prkdcscid/NcrCrl SCID mice immediately after induction of hind limb ischemia. Laser Doppler images show blood flow (BF) in the lower limbs of mice in prone position on day 14. (D) : BF recovery in the ischemic foot (calculated as a percentage ratio between ischemic foot BF and the contralateral foot) for each of the conditions. Statistical analysis shows significantly higher BF recovery in the EX‐FSTL3 treated mice at 14 days in comparison to controls; (Data is means ± SEM [ n = 3], *, p < .05). (E) : Sections of adductor muscles were stained with CD144 antibody for each condition. (F) : Quantified capillary density of CD144‐stained cells, expressed as capillary number per mm 2 (Data is means ± SEM [ n = 3], *, p < .05). (G) Quantified capillary density of CD144‐ and mCherry‐stained cells (Data is means ± SEM; **, p < .01; quantification from 10 random microscopic fields at ×40, scale: 50 μm). The data presented are representative or means (±SEM) of three independent experiments. Abbreviations: DAPI, 4′,6‐diamidino‐2‐phenylindole; ECs, endothelial cells; FSTL3, follistatin‐like 3.

Article Snippet: Antibodies against VE‐Cadherin (CD144) (ab33168 and STJ96234), VEGFR (ab9530), GAPDH (ab8245), OCT‐4 (ab19857 and STJ72238), KLF4 (ab72543) eNOS (ab76198), and FSTL3 (STJ112317) were purchased from Abcam, UK or St John's Laboratory, UK.

Techniques: In Vivo, Injection, Comparison, Control, Expressing, Staining, Saline, Muscles

Journal: Cancers

Article Title: The Thermal Dose of Photothermal Therapy Generates Differential Immunogenicity in Human Neuroblastoma Cells

doi: 10.3390/cancers14061447

Figure Lengend Snippet: PBNP-PTT generates a thermal dose window of immunogenic cell death in SH-SY5Y and LAN-1 cells in vitro. Three million ( A – C ) SH-SY5Y or ( D – F ) LAN-1 cells were exposed to various thermal doses using PBNP-PTT. After 24 h, cells were analyzed for ( A , D ) intracellular ATP, ( B , E ) HMGB1 release, and ( C , F ) surface calreticulin expression, represented as median fluorescence intensity (MFI). Inset values in the histograms denote the thermal dose. The extent of ICD as measured by its correlates is more pronounced in SH-SY5Y cells compared with LAN-1 cells. Ordinary one-way ANOVA was used to calculate significance between vehicle and different thermal doses and laser alone for HMB1 analysis. n = 2/group; * p < 0.03, ** p < 0.002, *** p < 0.0002, **** p < 0.0001.

Article Snippet: After 24 h incubation at 37 °C post-PBNP-PTT, cells were harvested and stained with Zombie Aqua Fixable viability dye (Biolegend, #423102), blocked with human TruStain Fc block (Biolegend, #422302), and stained with fluorescent antibodies against calreticulin (Abcam, #ab83220), CD80 (Biolegend, #305238), CD86 (Biolegend, #305428), PD-L1 (Biolegend, #329714), B7-H3 (Biolegend, #351010), HLA-ABC (Biolegend, #311432), HLA-DR (Biolegend, #307633), PVR (Biolegend, #337628), and GD2 (Biolegend, #357308).

Techniques: In Vitro, Expressing, Fluorescence

Journal: Cancers

Article Title: The Thermal Dose of Photothermal Therapy Generates Differential Immunogenicity in Human Neuroblastoma Cells

doi: 10.3390/cancers14061447

Figure Lengend Snippet: PBNP-PTT triggers greater immunophenotypic changes in MYCN-non-amplified SH-SY5Y cells than MYCN-amplified LAN-1 neuroblastoma cell line in vitro. SH-SY5Y (blue) and LAN-1 (green) cells were treated with varied thermal doses via PBNP-PTT and analyzed for ( A ) % live cells ( B ) intracellular ATP, ( C ) secreted HMGB1, and cell surface expression levels of ( D ) calreticulin, ( E ) CD80, ( F ) CD86, ( G ) PD-L1, ( H ) B7-H3, ( I ) HLA-ABC, ( J ) HLA-DR, ( K ) PVR, and ( L ) GD2. Data represent mean ± SD ( n = 2 independent samples).

Article Snippet: After 24 h incubation at 37 °C post-PBNP-PTT, cells were harvested and stained with Zombie Aqua Fixable viability dye (Biolegend, #423102), blocked with human TruStain Fc block (Biolegend, #422302), and stained with fluorescent antibodies against calreticulin (Abcam, #ab83220), CD80 (Biolegend, #305238), CD86 (Biolegend, #305428), PD-L1 (Biolegend, #329714), B7-H3 (Biolegend, #351010), HLA-ABC (Biolegend, #311432), HLA-DR (Biolegend, #307633), PVR (Biolegend, #337628), and GD2 (Biolegend, #357308).

Techniques: Amplification, In Vitro, Expressing

Journal: International Journal of Clinical and Experimental Pathology

Article Title: MAPK/ERK signal pathway involved expression of COX-2 and VEGF by IL-1? induced in human endometriosis stromal cells in vitro

doi:

Figure Lengend Snippet: Expression of COX-1 by 4 cytokines in endometriosis ESC by Western Blotting in vitro. Expression of COX-1 was expressed in three ESC, the normal was higher than the eutopic or ectopic, and there was statistical difference (P<0.05). The eutopic and ectopic had no statistical difference (P>0.05). And the expression of COX-1 had no statistical differences after 4 kinds CK (IL-1β, TNF-α, IFN-γ and M-CSF) was added (P>0.05).

Article Snippet: Macrophage colony stimulating factor (MCSF) and vascular endothelial growth factor (VEGF) development ELISA Kit were from PeproTech, Inc (Rocky Hill, NJ, USA), protein assay kit was from Bio-RAD (Hercules, CA, USA), antibody against COX-2 was from Cayman Chemicals (Ann arbor, Mi, USA), antibody against COX-1 was from Santa Cruz Biotechnology (Santa Cruz, CA, USA), the second antibody, a horseradish peroxidase-conjugated anti-mouse antibody and ECL plus western blotting detection system were obtained from Amersham (Piscataway, NJ, USA).

Techniques: Expressing, Western Blot, In Vitro

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