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: International immunopharmacology

Article Title: Inhibiting the SARM1-NAD + axis reduces oxidative stress-induced damage to retinal and nerve cells.

doi: 10.1016/j.intimp.2024.112193

Figure Lengend Snippet: Fig. 6. DSRM ameliorated oxidative stress-induced neurite injury in N2a cells. (A-C) GOx inhibited N2a cell proliferation and neurite growth in the RA-induced N2a cell differentiation model (magnification 400 × ); (D-E) Immunocytochemistry demonstrated that 10 μM DSRM pre-treatment for 2 h enhanced β3-tubulin expression induced by 7 mU/mL GOx for 12 h in N2a cells (scale bar: 50 μm). N ≥3, **p < 0.01, ****p < 0.0001.

Article Snippet: After culturing and treating the cells in 24-well plates and fixing and permeabilizing them with methanol at -20 ◦C for 15 min, the cells were then washed 3 times with PBS, and then incubated in containment buffer (5 % donkey serum for containment) for 20 min, and then incubated with anti-GSDMD primary antibody (1:500, Abclonal, Wuhan, China, Cat#A20197), anti-β3-tubulin primary antibody (1:1000, CST, USA, Cat#5568S) was incubated at 4 ◦C overnight.

Techniques: Cell Differentiation, Immunocytochemistry, Expressing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Vital functions of corticotropin-releasing factor (CRF) pathways in maintenance and regulation of energy homeostasis

doi: 10.1073/pnas.0511320103

Figure Lengend Snippet: Increased basal IBAT temperatures in 3-mo-old CRFR2-deficient (KO) mice compared with WT mice. (A) Basal and stimulated IBAT temperatures in mice were measured by TSA and displayed as a color-coded image. Computer-generated region of interest circles (green) are shown above the BAT region. (B) BAT thermogenesis was stimulated with the β3 agonist, BRL, and BAT temperatures were measured 15, 30, and 45 min after treatment. IBAT temperatures were significantly higher in vehicle-treated KO than vehicle-treated WT mice. BRL treatment significantly elevated WT IBAT temperatures compared with vehicle WT temperatures. KO mice showed no significant additional response to BRL treatment. Data are mean ± SEM; ∗∗, P < 0.01. (C) Thermogenic response to BRL treatment in IBAT temperatures in aged KO mice compared with WT mice measured by TSA. BAT thermogenesis was stimulated with the β3 agonist, BRL, and BAT temperatures were measured 15, 30, and 45 min after treatment. IBAT temperatures were significantly higher in BRL-treated aged KO mice than vehicle-treated KO mice. BRL treatment did not elevate WT IBAT temperatures compared with vehicle WT temperatures. Data are mean ± SEM; ∗∗, P < 0.01.

Article Snippet: The following primary antibodies were used: β3 receptor at 1:200 (Santa Cruz Biotechnology), TH at 1:1,000 (Pel-Freez Biologicals, Rogers, AR), pCREB (Ser-133) at 1:500 (Cell Signaling Technology, Beverly, MA), CREB at 1:1,000 (Santa Cruz Biotechnology), and β-actin at 1:1,000 (Sigma-Aldrich). β3, TH, and CREB levels were normalized to β-actin. pCREB was normalized to total CREB.

Techniques: Generated

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Vital functions of corticotropin-releasing factor (CRF) pathways in maintenance and regulation of energy homeostasis

doi: 10.1073/pnas.0511320103

Figure Lengend Snippet: Biochemical analyses of SNS pathway components in young and aged WT and KO mice. (A and B) Expression of TH in BAT was not different between genotypes of young (A) or aged (B) adult mice. #, TH levels decreased with age in WT mice (young, 4.24 ± 0.5 OD; aged, 2.29 ± 0.1 OD) but not KO mice (young, 2.43 ± 0.2 OD; aged, 2.22 ± 0.3 OD). (C and D) β3 levels were not different between genotypes or ages of young (C) or aged (D) WT and KO mice. (E) Expression of activated BAT pCREB levels was significantly elevated in young KO mice compared with WT mice. (F) No differences were found between aged WT and KO mice for pCREB expression. All OD analyses were conducted for within gel comparisons only. Data are mean ± SEM; ∗, P < 0.05.

Article Snippet: The following primary antibodies were used: β3 receptor at 1:200 (Santa Cruz Biotechnology), TH at 1:1,000 (Pel-Freez Biologicals, Rogers, AR), pCREB (Ser-133) at 1:500 (Cell Signaling Technology, Beverly, MA), CREB at 1:1,000 (Santa Cruz Biotechnology), and β-actin at 1:1,000 (Sigma-Aldrich). β3, TH, and CREB levels were normalized to β-actin. pCREB was normalized to total CREB.

Techniques: Expressing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Vital functions of corticotropin-releasing factor (CRF) pathways in maintenance and regulation of energy homeostasis

doi: 10.1073/pnas.0511320103

Figure Lengend Snippet: RER was significantly lower in KO compared with WT mice after high-fat diet challenge that was normalized after NBI treatment. This effect on RER in KO mice was reversed with CRFR1 antagonist NBI-27914 treatment. (A) VO2 for WT and KO mice with placebo or NBI pellets showing an increase in O2 consumption in KO mice. Neither WT nor KO mice show a significant change with NBI treatment. (B) VCO2 for WT and KO mice with placebo or NBI pellets showing an increase in CO2 production in KO mice. Neither WT nor KO mice showed a significant change with NBI treatment. (C) Placebo-treated mice showed a reduction in RER in KO mice compared with placebo WT mice, indicative of elevated basal metabolic rate in the KO mice. KO mice treated with a CRFR1 antagonist, NBI-27914, showed a significant increase in RER. NBI treatment produced no effect on WT RER. Data are mean ± SEM; ∗, P < 0.05.

Article Snippet: The following primary antibodies were used: β3 receptor at 1:200 (Santa Cruz Biotechnology), TH at 1:1,000 (Pel-Freez Biologicals, Rogers, AR), pCREB (Ser-133) at 1:500 (Cell Signaling Technology, Beverly, MA), CREB at 1:1,000 (Santa Cruz Biotechnology), and β-actin at 1:1,000 (Sigma-Aldrich). β3, TH, and CREB levels were normalized to β-actin. pCREB was normalized to total CREB.

Techniques: Produced

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Vital functions of corticotropin-releasing factor (CRF) pathways in maintenance and regulation of energy homeostasis

doi: 10.1073/pnas.0511320103

Figure Lengend Snippet: KO mice show a significant temperature preference for a warm environment. WT and KO mice were examined for time spent on each side of a temperature preference box. KO mice spent 3-fold the time on the warm compared with the RT side of the box. WT mice showed no difference in time spent on either warm or RT side (overall side ratio = 0.98). Data are mean ± SEM; ∗∗, P < 0.01.

Article Snippet: The following primary antibodies were used: β3 receptor at 1:200 (Santa Cruz Biotechnology), TH at 1:1,000 (Pel-Freez Biologicals, Rogers, AR), pCREB (Ser-133) at 1:500 (Cell Signaling Technology, Beverly, MA), CREB at 1:1,000 (Santa Cruz Biotechnology), and β-actin at 1:1,000 (Sigma-Aldrich). β3, TH, and CREB levels were normalized to β-actin. pCREB was normalized to total CREB.

Techniques:

Journal:

Article Title: c-Myc controls the balance between hematopoietic stem cell self-renewal and differentiation

doi: 10.1101/gad.313104

Figure Lengend Snippet: Expression of cell-adhesion molecules on KLS-HSCs

Article Snippet: Anti-E-cadherin-FITC (36), CD9 (KMC8)-biotin; CD29 (Ha2/5)-biotin and purified; CD49a (α1 integrin, HA3/18)-biotin; CD49e (α5 integrin, 5H10-27)-biotin; CD61 (β3 integrin, 2C9.G2)-biotin; CD104 (β4 integrin, 346.11A)-biotin; Sca-1 (2B8)-biotin; β7 integrin (M293)-biotin; CD49f (α6 integrin, GoH3)-PE; and CD184 (CXCR4, 2B11)-PE were purchased from BD Biosciences.

Techniques: Expressing

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