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: Journal of Translational Medicine

Article Title: AQP5-1364A/C polymorphism and the AQP5 expression influence sepsis survival and immune cell migration: a prospective laboratory and patient study

doi: 10.1186/s12967-016-1079-2

Figure Lengend Snippet: Western blot and migration assay of transfected Jurkat-cells and AQP5-immunostaining of neutrophils obtained from healthy donors a Western blot from whole cell lysates of Jurkat-cells ( lane u untransfected cells, lane c transfected with control plasmid, lanes 1 – 3 AQP5_pReceiver transfected Jurkat cells) with AQP5 and ACTB antibody; As indicated by the stronger band in lanes 1 – 3 , Jurkat cells could successfully be stable transfected; b Migration assay performed with cell culture inserts utilizing transfected Jurkat cells stimulated with SDF-1α and counted after 22 h (n = 6, 6); AQP5 overexpressing (AQP5_pRec) cells showed increased migration (p = 0.009) compared to control cells (pRec). c Representative examples of immunostaining with AQP5 antibody of neutrophils obtained from two healthy AA genotype carriers showing a considerable amount of AQP5 protein ( brown precipitate ). In neutrophils of two CC genotype carriers no clear AQP5 protein expression is detected. Every staining was performed in duplicate and eight random photographs were taken from each slide, 40× magnification

Article Snippet: Stable transfection with Jurkat T-cells was performed using an AQP5_pReceiver EX-T1015-M09 vector (Human Full ORF-Clone, NM-001651, pReceiver-M09) or a pReceiverM09 (GeneCopeia, Rockville, MD, USA).

Techniques: Western Blot, Migration, Transfection, Immunostaining, Control, Plasmid Preparation, Cell Culture, Expressing, Staining

Journal:

Article Title: Uncouplers of Oxidative Phosphorylation Can Enhance a Fas Death Signal

doi:

Figure Lengend Snippet: CCCP and DNP enhance the Fas apoptosis signal in Jurkat cells. Jurkat cells (2 × 105 per well) were incubated for 6 h in 96-well plates in 200 μl of complete medium or with various concentrations of CCCP (top panels) or DNP (bottom panels) as indicated either without (○) or with (●) anti-Fas MAb (100 ng/ml). Aliquots of the cultures were then assayed for apoptotic changes either morphologically (left panels) or after staining with propidium iodide for DNA content by flow cytometry (right panels). The experiments for these titration curves were performed three times with very similar results.

Article Snippet: The following cell lines were used in this study: Jurkat human T-cell leukemia (ATCC) cells, Jurkat cells overexpressing human Bcl-2 under the control of the EF1α promoter ( 36 ), the T-cell line CEM (provided by Marcus Peter, DKFZ, Heidelberg, Germany), and CEM CrmA ( 10a ) and the lymphoblastoid cell line SKW6 (both provided by Andreas Strasser, WEHI, Melbourne, Australia).

Techniques: Incubation, Staining, Flow Cytometry, Titration

Journal:

Article Title: Uncouplers of Oxidative Phosphorylation Can Enhance a Fas Death Signal

doi:

Figure Lengend Snippet: Morphology of Jurkat cells treated with anti-Fas–DNP. Jurkat cells (106 in 1 ml of complete culture medium in a 6-well plate) were incubated with the indicated stimuli (DNP was used at 1 mM, and anti-Fas [α-fas] was used at 100 ng/ml). Pictures were obtained after 6 h. A very similar effect was observed more than 20 times.

Article Snippet: The following cell lines were used in this study: Jurkat human T-cell leukemia (ATCC) cells, Jurkat cells overexpressing human Bcl-2 under the control of the EF1α promoter ( 36 ), the T-cell line CEM (provided by Marcus Peter, DKFZ, Heidelberg, Germany), and CEM CrmA ( 10a ) and the lymphoblastoid cell line SKW6 (both provided by Andreas Strasser, WEHI, Melbourne, Australia).

Techniques: Incubation

Journal:

Article Title: Uncouplers of Oxidative Phosphorylation Can Enhance a Fas Death Signal

doi:

Figure Lengend Snippet: CCCP and DNP enhance the Fas apoptosis signal in CEM cells. CEM cells (2 × 105 per well) were incubated for 3 h in 96-well plates in 200 μl of complete medium or with various concentrations of CCCP (top panels) or DNP (bottom panels) as indicated either without (○) or with (●) Anti-Fas MAb (100 ng/ml). Aliquots of the cultures were then assayed for apoptotic changes either morphologically (left panels) or after staining with propidium iodide for DNA content by flow cytometry (right panels). Note that the concentrations of CCCP required are lower than for Jurkat cells. Experiments for these titration curves were performed three times with very similar results.

Article Snippet: The following cell lines were used in this study: Jurkat human T-cell leukemia (ATCC) cells, Jurkat cells overexpressing human Bcl-2 under the control of the EF1α promoter ( 36 ), the T-cell line CEM (provided by Marcus Peter, DKFZ, Heidelberg, Germany), and CEM CrmA ( 10a ) and the lymphoblastoid cell line SKW6 (both provided by Andreas Strasser, WEHI, Melbourne, Australia).

Techniques: Incubation, Staining, Flow Cytometry, Titration

Journal:

Article Title: Uncouplers of Oxidative Phosphorylation Can Enhance a Fas Death Signal

doi:

Figure Lengend Snippet: CCCP and DNP do not alter the susceptibility of Jurkat cells to apoptosis induced by staurosporine. Jurkat cells (2 × 105 per well) were incubated for 6 h in 96-well plates in 200 μl of complete medium or with various concentrations of CCCP (top panels) or DNP (bottom panels) as indicated either without (○) or with (●) staurosporine (1 μM). Aliquots of the cultures were then assayed for apoptotic changes either morphologically (left panels) or after staining with propidium iodide for DNA content by flow cytometry (right panels). The experiments for these titration curves were performed three times with very similar results.

Article Snippet: The following cell lines were used in this study: Jurkat human T-cell leukemia (ATCC) cells, Jurkat cells overexpressing human Bcl-2 under the control of the EF1α promoter ( 36 ), the T-cell line CEM (provided by Marcus Peter, DKFZ, Heidelberg, Germany), and CEM CrmA ( 10a ) and the lymphoblastoid cell line SKW6 (both provided by Andreas Strasser, WEHI, Melbourne, Australia).

Techniques: Incubation, Staining, Flow Cytometry, Titration

Journal:

Article Title: Uncouplers of Oxidative Phosphorylation Can Enhance a Fas Death Signal

doi:

Figure Lengend Snippet: (a) CCCP and DNP enhance the Fas apoptosis signal in Jurkat Bcl-2 cells. Jurkat Bcl-2 cells (2 × 105 per well) were incubated for 7 h in 96-well plates in 200 μl of complete medium or with various concentrations of CCCP (top panels) or DNP (bottom panels) as indicated either without (○) or with (●) anti-Fas MAb (100 ng/ml). Aliquots of the cultures were then assayed for apoptotic changes either morphologically (left panels) or after staining with propidium iodide for DNA content by flow cytometry (right panels). In some experiments the titration ranges were different (as shown). The experiments for these titration curves were performed twice with similar results. More than three additional experiments with CCCP at 100 μM and DNP at 1 mM were also done; in these experiments CCCP and DNP showed a similar enhancing effect. (b) High-level Bcl-2 protects Jurkat cells against staurosporine-induced apoptosis. A total of 2 × 105 Jurkat cells (solid symbols) or Jurkat Bcl-2 cells (open symbols) per well were incubated for 7 h in 96-well plates in 200 μl of complete medium or with various concentrations of staurosporine as indicated. Aliquots of the cultures were then assayed for apoptotic changes either morphologically (circles) or after staining with propidium iodide for DNA content by flow cytometry (squares).

Article Snippet: The following cell lines were used in this study: Jurkat human T-cell leukemia (ATCC) cells, Jurkat cells overexpressing human Bcl-2 under the control of the EF1α promoter ( 36 ), the T-cell line CEM (provided by Marcus Peter, DKFZ, Heidelberg, Germany), and CEM CrmA ( 10a ) and the lymphoblastoid cell line SKW6 (both provided by Andreas Strasser, WEHI, Melbourne, Australia).

Techniques: Incubation, Staining, Flow Cytometry, Titration

Journal:

Article Title: Uncouplers of Oxidative Phosphorylation Can Enhance a Fas Death Signal

doi:

Figure Lengend Snippet: (a) CCCP and DNP enhance DEVD-cleaving activity in extracts from cells treated with anti-Fas. Jurkat cells (106 per time point) were incubated in 1 ml of complete medium containing no stimulus (○, at left), CCCP (200 μM; ○), DNP (2 mM; □), anti-Fas MAb (100 ng/ml; ▴), anti-Fas MAb and CCCP (●), or anti-Fas MAb and DNP (■). The agents were added at the times indicated prior to extraction, and all samples were harvested at the same time. Extracts were assayed in triplicate to determine the DEVD-AMC cleaving activity, and values are given as the mean/3 × the SEM. (For details of the extraction and assay for enzyme activity, see Materials and Methods). This kinetics experiment was done at least four times with all of the stimuli used at various concentrations of CCCP and DNP. A similar enhancing effect was seen in all experiments. (b) DEVD-cleaving activity is blocked by Z-VAD-fmk. Extracts were prepared from 106 unstimulated Jurkat cells (triangle) or Jurkat cells stimulated either with anti-Fas (100 ng/ml) and DNP (1 mM; circles) or with staurosporine (1 μM; squares) for the times indicated. Cells were extracted, and the DEVD-cleaving activity was measured as described for panel a (open symbols). Z-VAD-fmk (final concentration, 25 μM) was added 5 min before the addition of substrate to one aliquot of extract (colosed symbols). This experiment was performed twice with very similar results.

Article Snippet: The following cell lines were used in this study: Jurkat human T-cell leukemia (ATCC) cells, Jurkat cells overexpressing human Bcl-2 under the control of the EF1α promoter ( 36 ), the T-cell line CEM (provided by Marcus Peter, DKFZ, Heidelberg, Germany), and CEM CrmA ( 10a ) and the lymphoblastoid cell line SKW6 (both provided by Andreas Strasser, WEHI, Melbourne, Australia).

Techniques: Activity Assay, Incubation, Extraction, Concentration Assay

Journal:

Article Title: Uncouplers of Oxidative Phosphorylation Can Enhance a Fas Death Signal

doi:

Figure Lengend Snippet: Staining for ΔΨm in Jurkat cells after treatment with anti-Fas and uncouplers of oxidative phosphorylation. (a) Appearance of a rhodamine-123-high-staining population. Jurkat cells (106 per sample) were treated for 6 h with the agents indicated (anti-Fas, 100 ng/ml; CCCP, 100 μM; DNP, 1 mM). Cells were then stained and analyzed as described in Materials and Methods. The curves show untreated (control) cells as dotted lines and treated cells as solid lines. The mean fluorescence values for cells in the top panel were as follows: untreated, 25.1; CCCP treated, 21.6; and DNP treated, 17.8. This experiment was done at least five times with each agent with very similar results. (b) The appearance of a rhodamine-123-high-staining population is blocked by Z-VAD-fmk. Jurkat cells were treated as for panel a. Cells were treated with anti-Fas–DNP with or without Z-VAD-fmk (50 μM) as indicated. Cells were stained and analyzed as described above. Results are shown for control (untreated) cells (dotted lines) and treated cells (solid lines) plotted in each diagram. Treatment with Z-VAD-fmk alone did not change the rhodamine-123 staining (not shown). These experiments were done four times with very similar results. (c) Changes in JC-1 staining after treatment. Cells were treated as for panel a and were stained and analyzed as described in Materials and Methods. Results are shown for untreated cells (dotted lines) or cells treated with the indicated agents (solid lines). The treatment lasted 6 h as for panel a. This experiment was done three times with similar results.

Article Snippet: The following cell lines were used in this study: Jurkat human T-cell leukemia (ATCC) cells, Jurkat cells overexpressing human Bcl-2 under the control of the EF1α promoter ( 36 ), the T-cell line CEM (provided by Marcus Peter, DKFZ, Heidelberg, Germany), and CEM CrmA ( 10a ) and the lymphoblastoid cell line SKW6 (both provided by Andreas Strasser, WEHI, Melbourne, Australia).

Techniques: Staining, Phospho-proteomics, Control, Fluorescence

Journal:

Article Title: Uncouplers of Oxidative Phosphorylation Can Enhance a Fas Death Signal

doi:

Figure Lengend Snippet: Enhancement of the anti-Fas signal by DNP occurs in the absence of detectable cytochrome c redistribution. (a) Jurkat cells (4 × 107/sample) were treated with the agents indicated as described above. After 2 h, cells were collected by centrifugation and homogenized as described in Materials and Methods. Supernatants (containing cytosol and light membranes) and pellets (containing mitochondria) from the 10,000 × g centrifugation step were separated by SDS-PAGE and analyzed by Western blotting. Separate blots were probed with MAb to cytochrome oxidase subunit I (COX, top) and cytochrome c (bottom). These results are representative of five independent experiments. (b) Jurkat cells (2 × 107/sample) were treated for 2.5 h as indicated (fas/DNP, anti-Fas MAb [100 ng/ml] and DNP [1 mM]; stauro, staurosporine [1 μM]). Fractions were prepared and analyzed as described above.

Article Snippet: The following cell lines were used in this study: Jurkat human T-cell leukemia (ATCC) cells, Jurkat cells overexpressing human Bcl-2 under the control of the EF1α promoter ( 36 ), the T-cell line CEM (provided by Marcus Peter, DKFZ, Heidelberg, Germany), and CEM CrmA ( 10a ) and the lymphoblastoid cell line SKW6 (both provided by Andreas Strasser, WEHI, Melbourne, Australia).

Techniques: Centrifugation, SDS Page, Western Blot

Journal:

Article Title: Uncouplers of Oxidative Phosphorylation Can Enhance a Fas Death Signal

doi:

Figure Lengend Snippet: Addition of cytochrome c is required for the appearance of pro-caspase 9-processing activity in cytosolic extracts from Jurkat cells treated with anti-Fas or anti-Fas–DNP. Cytosolic extracts were prepared from Jurkat cells which either had been left untreated or had been treated with anti-Fas (100 ng/ml), anti-Fas–DNP (1 mM), or staurosporine (1 μM) for 2 h as described in Materials and Methods. Aliquots (100 μg) were incubated in a final volume of 60 μl of S-100 buffer with 1 μl of in vitro-translated pro-caspase 9 for 3 h at 37°C either alone (left), in the presence of 1 mM dATP (middle), or in the presence of 1 mM dATP and 1 μg of cytochrome c (right). Reactions were then boiled in Laemmli buffer and subjected to SDS-PAGE. Gels were dried, and caspase-9 was visualized by autoradiography. Arrow, intact pro-caspase 9; arrowhead, processed fragments (ca. 35 kDa). Very similar results were obtained in four experiments with three independent sets of extracts.

Article Snippet: The following cell lines were used in this study: Jurkat human T-cell leukemia (ATCC) cells, Jurkat cells overexpressing human Bcl-2 under the control of the EF1α promoter ( 36 ), the T-cell line CEM (provided by Marcus Peter, DKFZ, Heidelberg, Germany), and CEM CrmA ( 10a ) and the lymphoblastoid cell line SKW6 (both provided by Andreas Strasser, WEHI, Melbourne, Australia).

Techniques: Activity Assay, Incubation, In Vitro, SDS Page, Autoradiography

Journal:

Article Title: Uncouplers of Oxidative Phosphorylation Can Enhance a Fas Death Signal

doi:

Figure Lengend Snippet: Induction of DEVD-cleaving activity by cytochrome c in extracts from normal and apoptotic cells. Jurkat cells were treated as indicated (anti-Fas MAb, 100 ng/ml; DNP, 1 mM; staurosporine, 1 μM), and cytosolic extracts were prepared as described in Materials and Methods. Extracts (50 μg) were incubated in 40-μl reaction mixtures for 90 min at 37°C. DEVD-cleaving activity was then measured in triplicates of 10 μl from each mixture as described in the text. Results are given as the mean/3 × the SEM. (a) Extracts were prepared after 2 h of the indicated treatment and incubated either alone (solid columns), with 1 mM dATP (open columns), or with 1 mM dATP and 0.5 μg of cytochrome c (hatched columns). (b) Cells were either left untreated (time zero) or were treated with a combination of anti-Fas MAb and DNP for the time periods indicated. Extracts were prepared and incubated either alone (solid columns) or with 1 mM dATP and 0.5 μg of cytochrome c (open columns). (c) Cells were either left untreated (time zero) or were treated with staurosporine for 1 or 2 h as indicated. Extracts were prepared and incubated alone (solid columns), with 1 mM dATP (open columns), or with 1 mM dATP and 0.5 μg of cytochrome c (hatched columns).

Article Snippet: The following cell lines were used in this study: Jurkat human T-cell leukemia (ATCC) cells, Jurkat cells overexpressing human Bcl-2 under the control of the EF1α promoter ( 36 ), the T-cell line CEM (provided by Marcus Peter, DKFZ, Heidelberg, Germany), and CEM CrmA ( 10a ) and the lymphoblastoid cell line SKW6 (both provided by Andreas Strasser, WEHI, Melbourne, Australia).

Techniques: Activity Assay, Incubation