Journal: Nature Chemical Biology

Article Title: 2′-Deoxyadenosine 5′-diphosphoribose is an endogenous TRPM2 superagonist

doi: 10.1038/nchembio.2415

Figure Lengend Snippet: Figure 4 | NMNAT-2 and CD38 synthesize 2′-deoxy-ADPR in vitro. (a) HPLC analysis of the reaction products of β-NMN and 2′-deoxy-ATP incubated with or without recombinant hNMNAT-2. 2′-deoxy-AMP and 2′-deoxy-ADP were impurities contained in commercial 2′-deoxy-ATP. (b) A saturation plot for ATP and 2′-deoxy-ATP as substrates of NMNAT-2; β-NMN was used at 290 μmol/L for both ATP and 2′-deoxy-ATP. The initial reaction rate was calculated from the amount of product formed, as determined by HPLC. Data shown are the mean ± s.d. (n = 3 independent experiments with the concentration range shown for ATP; for 2′-deoxy-ATP, three additional experiments were performed for 3 mM, 5 mM, 7 mM and 9 mM, so n = 6 for 3 mM and 9 mM)). Data for ATP were fitted to a Michaelis–Menten model. Data up to 3 mmol/L 2′-deoxy-ATP were fitted to a Hill model (black curve and inset). At higher concentrations the reaction rate dropped rapidly, indicating substrate inhibition (dashed lines). (c) Recombinant human CD38 hydrolyzed 2′-deoxy-NAD (from a) to yield the TRPM2 agonist 2′-deoxy-ADPR. (d) CD38 activity as determined by incubation of Jurkat cells with 1,N6-etheno- NAD and HPLC analysis of the product 1,N6-etheno-ADPR at different time points. To analyze CD38 activity in the type III orientation (red symbols in the schematic in the upper right), we inhibited CD38 activity on the cell surface with covalently bound araF-NAD (blue and gray symbols along the outside of the circle). To obtain access to type III CD38, we selectively permeabilized the plasma membrane with saponin. After determination of the type III CD38 activity, cells were incubated with araF-NAD again (red and gray symbols along the inside of the circle). Data in bar graphs are the mean and s.e.m.; the number of experiments is indicated for each bar. For conditions tested in a one-way ANOVA for repeated measurements, with Tukey’s correction for multiple testing, the corrected P values are shown. Data in a and c are representative of results from three independent experiments. Rt, retention time.

Article Snippet: The cells were authenticated as Jurkat cells by short tandem repeat profiling and tested negative for contamination with rodent cells (DSMZ service for the authentication of human cell lines).

Techniques: In Vitro, Incubation, Recombinant, Concentration Assay, Inhibition, Activity Assay, Clinical Proteomics, Membrane

Journal: Nature Chemical Biology

Article Title: 2′-Deoxyadenosine 5′-diphosphoribose is an endogenous TRPM2 superagonist

doi: 10.1038/nchembio.2415

Figure Lengend Snippet: Figure 5 | The increase in 2′-deoxy-ADPR in Jurkat cells exposed to hydrogen peroxide depends on CD38. Wild-type and CD38−/− Jurkat cells were either exposed to 100 μmol/L H2O2 for 5 min or left unstimulated (Unstim.). (a,d) Deproteinized extracts from cell samples were separated by RP-HPLC; the graphs show the absorbance of fractions that coeluted with authentic ADPR, NAD, 2′-deoxy-ADPR or 2′-deoxy-NAD. (b,c,e,f) Representative chromatograms showing the absorbance of fractions from the prefractionation that were subjected to a second level of RP-HPLC. To correctly assign peaks, we split each sample and spiked one half with the respective nucleotide. Because we could not achieve baseline separation for 2′-deoxy-ADPR, we did not analyze it quantitatively; however, the peak of 2′-deoxy-ADPR clearly increased after hydrogen peroxide stimulation. In the chromatograms from CD38−/− cells (e and f), the peak for 2′-deoxy-ADPR was not detected regardless of stimulation. (g) Quantitative analysis of the effect of H2O2 on intracellular 2′-deoxy-NAD. Independent experiments were initiated on three separate days over the course of one month, with each experiment consisting of multiple parallel cell preparations. We obtained 2–6 data points per experimental day. Results from single experiments are indicated by solid data points (horizontal lines indicate means); data from experiments on the same day are shown in the same shade of gray. Data are normally distributed in each group. Analysis by one-way ANOVA did not reveal significant differences (P = 0.074). AU, absorbance units.

Article Snippet: The cells were authenticated as Jurkat cells by short tandem repeat profiling and tested negative for contamination with rodent cells (DSMZ service for the authentication of human cell lines).

Techniques:

Journal: Journal of Biological Chemistry

Article Title: Modulation of T Cell Cytokine Production by Interferon Regulatory Factor-4

doi: 10.1074/jbc.m205895200

Figure Lengend Snippet: FIG. 1. Early activation events in IRF-4-transfected cells. A, whole cell extracts were prepared from Jurkat cells stably transfected with either a control or an IRF-4 expression vector, electrophoresed on a 7% SDS-polyacrylamide gel, and then analyzed by Western blotting using an anti-IRF-4 antibody (upper panel). The blot was later stripped and reprobed with a -actin antibody (lower panel) to ensure for equal loading. Extracts from untransfected Jurkat cells and HUT 78 served, respectively, as negative and positive controls. B, Jurkat-transfected cells were either left unstimulated or were stimulated with PMA (50 ng/ml) and ionomycin (1 M) for 24 h. The cells were then harvested and stained with either a phycoerythrin-labeled anti-CD69 (upper panel) or a phycoerythrin-labeled anti-CD25 antibody (lower panel) and analyzed by flow cytometry. Filled histograms represent unstimulated cells, whereas empty histograms represent cells stimulated with PMA and ionomycin. Left panel, vector transfectants; right panel, IRF-4 transfec- tants. Not shown is staining with an isotype-matched control, which did not reveal any significant differences between control and IRF-4 transfectants.

Article Snippet: Cell Lines and Cultures—The Jurkat (human T cell leukemia) cell line was obtained from American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Activation Assay, Transfection, Stable Transfection, Control, Expressing, Plasmid Preparation, Western Blot, Staining, Labeling, Flow Cytometry

Journal: Journal of Biological Chemistry

Article Title: Modulation of T Cell Cytokine Production by Interferon Regulatory Factor-4

doi: 10.1074/jbc.m205895200

Figure Lengend Snippet: FIG. 4. IRF-4 transactivates the human IL-2 and IL-4 promoters. Control and IRF-4 Jurkat-transfected cells were transiently transfected with a luciferase reporter construct driven either by the human IL-2 promoter (left panel) or the human IL-4 promoter (right panel). The transfected cells were equally split into two 2-ml aliquots and then incubated for 4 h in the presence or absence of PMA (50 ng/ml) and ionomycin (1 M). The data are presented relative to the activity of the reporter construct in unstimulated control cells, which was set to 1.0, as indicated in each experiment. Results show the mean S.E. of five (for the IL-2 promoter) and six (for the IL-4 promoter) independent experiments.

Article Snippet: Cell Lines and Cultures—The Jurkat (human T cell leukemia) cell line was obtained from American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Control, Transfection, Luciferase, Construct, Incubation, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: Modulation of T Cell Cytokine Production by Interferon Regulatory Factor-4

doi: 10.1074/jbc.m205895200

Figure Lengend Snippet: FIG. 6. IRF-4 can act as a transactivator of the P1-IRF element. Control and IRF-4 Jurkat cells were transfected with a luciferase re- porter construct driven by either an oligomerized P1-IRF wt or an oligomerized P1-IRFM3 element. The transfected cells were equally split into two 2-ml aliquots and then incubated for 4 h in the presence or absence of PMA (50 ng/ml) and ionomycin (1 M). The data are presented relative to the activity of the reporter construct in unstimu- lated control cells, which was set to 1.0, as indicated, in each experi- ment. Results show the mean S.E. of three independent experiments.

Article Snippet: Cell Lines and Cultures—The Jurkat (human T cell leukemia) cell line was obtained from American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Control, Transfection, Luciferase, Construct, Incubation, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: Modulation of T Cell Cytokine Production by Interferon Regulatory Factor-4

doi: 10.1074/jbc.m205895200

Figure Lengend Snippet: FIG. 7. IRF-4 cooperates with NFAT in driving T cell cytokine production. A, vector and IRF-4 Jurkat cells were co- transfected with a luciferase reporter con- struct driven by the human IL-4 promoter and either an NFATc1 expression vector or equivalent amounts of an empty vector. The transfected cells were equally split into two 2-ml aliquots and then incubated for 4 h in the presence or absence of PMA (50 ng/ml) and ionomycin (1 M). The data are presented relative to the activity of the reporter construct in vector control cells, which was set to 1.0, as indicated, in each experiment. Results show the mean S.E. of four independent experi- ments. B, control and IRF-4-transfected cells were either left unstimulated or stimulated with PMA and ionomycin as indicated in the legend to Fig. 2. Stimula- tions were conducted in the presence or absence of cyclosporin A (1 g/ml) or FK506 (10 ng/ml) as indicated. Superna- tants were then collected and analyzed for their cytokine content by ELISA. Data shown are representative of four inde- pendent experiments and performed on three independent sets of transfectants.

Article Snippet: Cell Lines and Cultures—The Jurkat (human T cell leukemia) cell line was obtained from American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Plasmid Preparation, Transfection, Luciferase, Expressing, Incubation, Activity Assay, Construct, Control, Enzyme-linked Immunosorbent Assay

Journal: Biology of reproduction

Article Title: Bezafibrate restores the inhibition of FSH-induced follicular development and steroidogenesis by tumor necrosis factor-alpha through peroxisome proliferator-activated receptor-gamma pathway in an in vitro mouse preantral follicle culture.

doi: 10.1095/biolreprod.111.090738

Figure Lengend Snippet: FIG. 4. Expression of PPAR subtypes in mouse preantral follicles is shown. mRNA (A) and protein (B) expression levels of PPAR subtypes in mouse preantral follicles are shown. Preantral follicles were mechanically isolated from mouse ovaries. One hundred preantral follicles were collected and subjected to RT-PCR and Western blot analysis. A) Total RNA was isolated from preantral follicles and various mouse tissues: liver, stomach, and adipose tissues. RT-PCR assay was performed to detect mRNA of PPARA (upper panel), PPARD (middle panel), and PPARG (lower panel). Positive controls for PPARA, PPARD, and PPARG were liver, stomach, and adipose tissue, respectively. GAPDH was used as an internal control. B) Protein expression levels of PPARA (upper panel), PPARD (middle panel), and PPARG (lower panel) were examined by Western blot analysis, using respective antibodies. a-Tubulin was used as an internal control. Positive controls for PPARA and PPARD were Hep G2 cell lysate and Jurkat nuclear extract, respectively (see Materials and Methods). Mouse adipose tissue was used as a positive control for PPARG. Experiments were repeated three times with consistent results, and representative results are shown. Presence (þ) or absence () of RT in the RT-PCR reaction is shown. P.C., positive control.

Article Snippet: Anti-PPARA antibody (catalog no. sc-9000), anti-PPARD antibody (catalog no. sc-74517), Hep G2 cell lysate (positive control for PPARA; catalog no. sc-2227), Jurkat nuclear extract (positive control for PPARD; catalog no. sc-2132), and anti-a-tubulin antibody (catalog no. sc-5286) for Western blotting were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Control, Positive Control