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