Journal:

Article Title: Defective T-Cell Activation Is Associated with Augmented Transforming Growth Factor ? Sensitivity in Mice with Mutations in the Sno Gene

doi: 10.1128/MCB.23.15.5446-5459.2003

Figure Lengend Snippet: T-cell proliferation assays examining functions of unprimed T cells show that Sno5Δ−/− and Snoex1−/− mutant T cells have a T-cell activation defect that is largely compensated for by addition of excess IL-2 or incubation with anti-TGF-β antibody. (A) Spleen cells from littermates of each genotype were seeded at a density of 500 × 103 responder cells per microwell in 96-well plates. The cells were incubated for 66 h at 37°C and then for a final 6 h with 1 μCi of [3H]thymidine and then harvested onto glass fiber filters to determine the [3H]thymidine incorporation. The numbers presented are kilocounts of [3H]thymidine per minute (background samples without stimulator were subtracted) in the average of triplicate wells from a representative experiment. The error bars indicate the calculated standard deviations. For T-cell receptor stimulation of splenocytes, 10 ng of 145-2C11 αCD3 allogeneic major histocompatibility complex anti-T-cell receptor (T-cell receptor) monoclonal antibody was preincubated in each well as indicated (aCD3). Additional antibody or cytokines were added as indicated (TGFb, TGF-β). Control wells had no αCD3 stimulator or other additions to the media and had very low proliferation. The genotypes were wild type (Sno+/+), Sno5Δ−/−, and Snoex1−/−. The asterisks indicate results that were statistically significantly (P < 0.05) different from the wild type. (B) Splenocytes were stimulated with PMA-ionomycin (PMA/io), with added cytokines as indicated. [3H]thymidine incorporation was measured on day 4 after plating. The genotypes were as in panel A. Representative experiments of 5 to 12 repetitions are shown in both panels.

Article Snippet: Anti-TGF-β antibody (MAB1835, clone 1D11 anti-TGF-β1, -β2, -β3; R&D Systems) was used at 2 μg/ml.

Techniques: Mutagenesis, Activation Assay, Incubation

Journal:

Article Title: Defective T-Cell Activation Is Associated with Augmented Transforming Growth Factor ? Sensitivity in Mice with Mutations in the Sno Gene

doi: 10.1128/MCB.23.15.5446-5459.2003

Figure Lengend Snippet: (A and B) Wild-type and mutant MEFs show different DNA synthetic rates (A) and Sno mutant cells are more sensitive to TGF-β (B). MEFs were isolated from litters of embryos derived from intercrossed mice that were either both wild type or both homozygous mutant. The genotype of each MEF preparation was verified by PCR; multiple preparations gave the same results in these experiments. Equal numbers of cells were plated in quadruplicate sets of microwells and untreated or treated with increasing concentrations of TGF-β or panspecific anti-TGF-β antibody at 2 μg/ml for 24 h. The cells were metabolically labeled for the final 3 h with 1 μCi of [3H]thymidine per well and harvested onto glass fiber filters to determine the [3H]thymidine incorporation. The asterisks above the control bars (panel A, control; panel B, 0.0 pM) indicate results that were statistically significantly (P < 0.05) different from the wild-type control. In panel A, the asterisks above the other bars indicate results that were statistically significantly (P < 0.05) different from the corresponding untreated control cells. In panel B, the asterisks at 100 pM TGF-β indicate significant (P < 0.02) difference from the wild type; the other asterisks indicate significant difference (P < 0.009) from the wild type. Incorporation into mutant cells was statistically significantly different from the wild type in the presence of anti-TGF-β antibody (Sno5Δ−/−, P < 0.013; Snoex1−/−, P < 0.001), whereas incorporation in mutants and the wild type was not significantly different in the presence of 5 pM TGF-β. The genotypes were wild type (Sno+/+), Sno5Δ−/−, and Snoex1−/−. Two independent experiments are shown with different absolute [3H]thymidine incorporation levels in the controls.

Article Snippet: Anti-TGF-β antibody (MAB1835, clone 1D11 anti-TGF-β1, -β2, -β3; R&D Systems) was used at 2 μg/ml.

Techniques: Mutagenesis, Isolation, Derivative Assay, Metabolic Labelling, Labeling

Journal:

Article Title: Defective T-Cell Activation Is Associated with Augmented Transforming Growth Factor ? Sensitivity in Mice with Mutations in the Sno Gene

doi: 10.1128/MCB.23.15.5446-5459.2003

Figure Lengend Snippet: MEFs from Snoex1−/− embryos show increased activity of the 3TP-lux and A3-lux TGF-β-responsive promoters, either with or without TGF-β supplementation of the cultures. (A) The activity of a TGF-β-responsive promoter element, 3TP-lux, was tested in transfected MEFs. The genotypes were wild type (Sno+/+), Sno5Δ−/−, and Snoex1−/−. “Control” indicates the level of luciferase activity of transfected 3TP-lux reporter alone. +TGF-b, TGF-β (100 pM) was added; +Sno, pCMV-SnoN expression construct was cotransfected. Sixty-millimeter-diameter dishes were transfected in triplicate, and the relative light units (RLU) emitted by the luciferase reporter were measured in duplicate in a luminometer. The error bars indicate the calculated standard deviations from each group of six values measured. The asterisks above the control bars indicate results that were statistically significantly (P < 0.05) different from the wild type. The asterisks above the other bars indicate results that were statistically significantly (P < 0.05) different from the corresponding untreated control cells. The results for the mutants were significantly different from the corresponding wild-type results under each condition (Snoex1−/−, P < 0.0007; Sno5Δ−/−, P < 0.034). (B) The increase with added TGF-β was plotted for Sno+/+ and Snoex1−/− cells from combined data from five experiments. Sno5Δ−/− cells had the same 2.7-fold increase as Sno+/+ cells and were not plotted. The asterisk indicates that the 3.2-fold increase in luciferase in the presence of TGF-β was statistically significantly (P ≤ 0.016) higher in Snoex1−/− cells than the 2.7-fold increase in the wild type. (C) The activity of a different TGF-β-responsive promoter element, A3-lux, was tested in wild-type and Snoex1−/− MEFs cotransfected with a FAST-2 expression vector. The asterisk indicates that the activity in the presence of TGF-β was statistically significantly higher in Snoex1−/− than in Sno+/+ MEFs (P < 0.005). (D) To confirm that MEFs of the three genotypes were transfected with similar efficiencies, a pSVβgal construct was transfected in parallel in the same experiment, and the dishes were stained and photographed. The transfection efficiencies were similar among the threegenotypes. The Snoex1−/− cells expressed lacZ from the knock-in construct, seen in the nuclear staining in the figure. The transfected pSVβgal gave cytoplasmic staining and was thus distinguishable from the nuclear-staining Snoex1−/− background.

Article Snippet: Anti-TGF-β antibody (MAB1835, clone 1D11 anti-TGF-β1, -β2, -β3; R&D Systems) was used at 2 μg/ml.

Techniques: Activity Assay, Transfection, Luciferase, Expressing, Construct, Plasmid Preparation, Staining, Knock-In

Journal:

Article Title: Defective T-Cell Activation Is Associated with Augmented Transforming Growth Factor ? Sensitivity in Mice with Mutations in the Sno Gene

doi: 10.1128/MCB.23.15.5446-5459.2003

Figure Lengend Snippet: Snoex1−/− MEFs show enhanced activation of endogenous JunB in response to TGF-β. (A) Real-time PCR measured levels of JunB endogenous mRNAs in wild-type and Sno mutant MEFs with (+TGFb) or without (−TGFb) incubation with 100 pM TGF-β for 2 h. The quantified levels calculated for each sample are presented in the histograms. Each sample was measured in quadruplicate and standardized against a dilution curve generated in the same experiment, using the same JunB primers and twofold serial dilutions of template (not shown). The genotypes are indicated below panel B. The asterisks indicate results that were statistically significantly (P < 0.015) different from the corresponding untreated cells. The difference in the presence of added TGF-β between Snoex1−/− and the wild type was significant (P < 0.018). (B) L7 ribosomal protein loading control real-time PCR results are presented as a histogram, showing that the samples contained comparable levels of RT-RNA; the profiles were not normalized. The error bars indicate the calculated standard deviations.

Article Snippet: Anti-TGF-β antibody (MAB1835, clone 1D11 anti-TGF-β1, -β2, -β3; R&D Systems) was used at 2 μg/ml.

Techniques: Activation Assay, Real-time Polymerase Chain Reaction, Mutagenesis, Incubation, Generated

Journal: PLoS ONE

Article Title: Adenovirus Gene Transfer to Amelogenesis Imperfecta Ameloblast-Like Cells

doi: 10.1371/journal.pone.0024281

Figure Lengend Snippet: Expression of α v β3 and α v β5 integrins ( A and B ) and heparin sulfate proteoglycans ( C–E ) in AI-WAm cell population and control cells was analyzed by flow cytometry ( A , C , E ) and IHC staining ( B and D ). Cells were incubated with primary anti-α v β3 or anti-α v β5 monoclonal antibodies for detection of corresponding integrin molecules or 10E4 antibody for detection of HSPG side chains (GAG) or anti-human syndecan 4 monoclonal antibody, followed by Alexa 488-conjugated secondary antibody. A and C , top charts: AI-WAm cells; middle charts: RD cells; bottom charts: A549 cells. For AI-WAm cells: P (αvβ3/αvβ5) = 0.75 ; P (Synd4/HSPG) = 0.29 ; For RD cells: P (Synd4/HSPG) = 0.67 ; for α v β3: P (RD/A549) = 0.38 ; for α v β5: P (AI-WAm/A549) = 0.23 ; for syndecan 4: P ( RD /A549) = 0.2 ; for all other differences P <0.05; E. HSPG Ab (10E4) specificity control sample: A549 cells were treated with heparitinase (10 U/ml) for 1 hr at 37°C to remove GAG side chains. Green arrow shows shift of the fluorescence intensity peak resulting from reduction in cell labeling with 10E4 antibody (MFI decrease). Other details are as in Fig. 2D . B and D , scale bars correspond to: 100 µm in top image panels (integrins/AI-WAm, 10× objective), 10 µm (insert, 60× objective) and 50 µm (40× objective) in all other panels. Insert shows syndecan 4 staining image (60×) of A549 cells, clearly demonstrating a polarized intracellular localization of the protein.

Article Snippet: After washes in PBS, the samples were blocked with 10% BSA (Sigma, St. Louis, MO) and incubated overnight at 4°C with one of the following primary antibodies: rabbit polyclonal anti-Amel (Sigma, St. Louis, MO), rabbit polyclonal anti-cytokeratin 14 (ab53115; Abcam, Cambridge, MA); goat polyclonal anti-Enam (C-18; Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-human syndecan 4 (Abcam, Cambridge, MA) at 1∶50 dilutions in 3% BSA/PBS or mouse monoclonal anti-hCAR antibody (clone RmcB, Millipore, Billerica, MA), mouse monoclonal anti-human HSPG/GAG 10E4 antibody (F58-10E4, Seikagaku Biobusiness Corp), mouse anti-human integrin α v β3 (LM609 clone) or α v β5 (P1F6 clone) monoclonal antibodies (500 µg/ml) (Millipore, Billerica, MA) at 1∶100 dilutions in 3% BSA/PBS at room temperature for 2 hrs.

Techniques: Expressing, Flow Cytometry, Immunohistochemistry, Incubation, Fluorescence, Labeling, Staining

Journal: PLoS ONE

Article Title: Adenovirus Gene Transfer to Amelogenesis Imperfecta Ameloblast-Like Cells

doi: 10.1371/journal.pone.0024281

Figure Lengend Snippet: A. Differential blocking of gene transfer to AI-WAm cells by integrins. Ad5 RGD shows the highest sensitivity to integrin blocking, while transduction with Ad5-pK7/RGD (G/L) is only partially inhibited. Ad5-pK7 (G/L) gene transfer shows no statistically significant inhibition by integrins. B. Blocking of AI-WAm gene transfer by modified vectors with heparin. Heparin shows a profound dose-dependent blocking effect on transduction with pK7-modified Ads, as opposed to RGD-modified vector. Gray bars (with % values on the top) show percentage of the residual gene transfer level (RLU) resulting from blocking relative to that of unblocked controls (100%) shown by black bar for each fiber-modified vector. All bars represent mean values with standard deviations. All differences were statistically significant except where indicated by asterisk and P values ( P >0.05) on the data bars.

Article Snippet: After washes in PBS, the samples were blocked with 10% BSA (Sigma, St. Louis, MO) and incubated overnight at 4°C with one of the following primary antibodies: rabbit polyclonal anti-Amel (Sigma, St. Louis, MO), rabbit polyclonal anti-cytokeratin 14 (ab53115; Abcam, Cambridge, MA); goat polyclonal anti-Enam (C-18; Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-human syndecan 4 (Abcam, Cambridge, MA) at 1∶50 dilutions in 3% BSA/PBS or mouse monoclonal anti-hCAR antibody (clone RmcB, Millipore, Billerica, MA), mouse monoclonal anti-human HSPG/GAG 10E4 antibody (F58-10E4, Seikagaku Biobusiness Corp), mouse anti-human integrin α v β3 (LM609 clone) or α v β5 (P1F6 clone) monoclonal antibodies (500 µg/ml) (Millipore, Billerica, MA) at 1∶100 dilutions in 3% BSA/PBS at room temperature for 2 hrs.

Techniques: Blocking Assay, Transduction, Inhibition, Modification, Plasmid Preparation

Journal: Nature

Article Title: Nasopharyngeal lymphatic plexus is a hub for cerebrospinal fluid drainage

doi: 10.1038/s41586-023-06899-4

Figure Lengend Snippet: a , Diagram of intracranial upstream lymphatic regions 1, 2 and 3, which drain through the NPLP en route to medial deep cervical lymphatics and dcLNs in the neck. Upstream lymphatic region 1 includes the lymphatics near the pituitary gland and cavernous sinus that drain to the NPLP. Upstream lymphatic region 2 includes the lymphatics in the anterior region of basolateral dura near the middle meningeal artery and petrosquamosal sinus (PSS) that course along the PPA to the NPLP. Upstream lymphatic region 3 includes lymphatics near the cribriform plate that drain to the lymphatics in the olfactory mucosa en route to the posterior nasal lymphatic plexus and NPLP. By contrast, the lymphatics in the posterior region of the basolateral dura around the sigmoid sinus do not drain to the NPLP but, instead, pass through the jugular foramen to lateral deep cervical lymphatics en route to dcLNs. Anatomical positions are indicated at the bottom left. b , Fluorescence image showing medial dcLVs, lateral dcLVs, lymphatic valves (green arrowheads) and TMR–dextran (red) in lymphatics deep in the neck of a Prox1-GFP mouse. The image was obtained 30 min after i.c. infusion of TMR–dextran (molecular mass, 10 kDa) at 1.0 μl min −1 for 3 min. Medial dcLVs connect to the NPLP, and lateral dcLVs connect to the basolateral dural lymphatics through the jugular foramen. Scale bar, 1 mm. Similar findings were obtained from n = 6 mice in three independent experiments. c–e , Immunofluorescence images of whole mounts showing the distributions of PROX1-dense, semi-lunar shaped lymphatic valves (green arrowheads) and αSMA + circular smooth muscle cells (SMCs, orange arrowheads) in the medial and lateral dcLVs. d , e , Magnified images of the regions indicated by the green boxes in c . Scale bar, 1 mm ( c ). Similar findings were obtained from n = 4 mice in two independent experiments. f , Immunofluorescence images of whole mounts showing a typical semi-lunar-shaped PROX1-dense, laminin-α5 high valve (yellow arrowheads) in a medial dcLV of a Prox1-GFP mouse. Scale bars, 200 μm. Similar findings were obtained from n = 4 mice in two independent experiments. g , Immunofluorescence images of whole mounts showing the distributions of β3-tubulin + axons (white arrowheads) and αSMA + circular smooth-muscle cells (red) along dcLVs. Scale bars, 200 μm. Similar findings were obtained from n = 4 mice in two independent experiments.

Article Snippet: The primary antibodies used were as follows: anti-LYVE1 (rabbit polyclonal, 11-034, Angiobio), anti-CD31 (hamster monoclonal, 2H8, MAB1398Z, Merck), anti-VE-cadherin (goat polyclonal, AF1002, R&D), anti-VEGFR3 (goat polyclonal, AF743, R&D), anti-αSMA-Cy3 (mouse monoclonal, 1A4, C6198, Sigma-Aldrich), anti-β3 tubulin (mouse monoclonal, 2G10, ab78078, Abcam), anti-FOXC2 (sheep polyclonal, AF6989, R&D), anti-LYVE1 (rabbit polyclonal, DP3500, OriGene), anti-collagen type IV (goat polyclonal, AB769, Merck), anti-laminin α5 (rabbit polyclonal, EWL004, Kerafast), anti-tyrosine hydroxylase (rabbit polyclonal, AB152, Merck), anti-vesicular acetylcholine transporter (goat polyclonal, ABN100, Merck), anti-phospho-tau (mouse monoclonal, AT8, MN1020, Thermo Fisher Scientific), anti-mannose receptor (CD206, rabbit polyclonal antibody, ab64693, Abcam), anti-PTX3 (rabbit polyclonal antibody, ALX-210-365-C050, Enzo Life Sciences).

Techniques: Fluorescence, Immunofluorescence